Bifunctional Polyester Synthase–Channel Driving Phosphorylated PHB–PHV Synthesis and Ion Conductance

Bifunctional Polyester Synthase–Channel Driving Phosphorylated PHB–PHV Synthesis and Ion Conductance | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Bifunctional Polyester Synthase–Channel Driving Phosphorylated PHB–PHV Synthesis and Ion Conductance Desouky A.M. Abd-El-Haleem This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6506981/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Microbial polyhydroxyalkanoates (PHAs) have traditionally been viewed as inert carbon reserves, yet emerging evidence implicates these polyesters in ion transport and stress response. Here, we identify and characterize ORF1 from Hanseniaspora valbyensis as the first genetically encoded polyester synthase–translocator that couples phosphorylated poly[(R)-3-hydroxybutyrate–3-hydroxyvalerate] (PHB–PHV) biosynthesis with vectorial export through a membrane pore. The 344residue ORF1 protein was heterologously expressed in Escherichia coli ΔphaC, and its product validated by TEM, FTIR, NMR and GC–MS. Six independent topology predictors (Phobius, PolyPhobius, MEMSATSVM, Philius, DeepTMHMM and MemBrain) and a TOPCONS2 metaanalysis converged on a central multihelix region (residues 159–222) as the membraneembedded core. AlphaFold2 modeling and PoreWalker analysis revealed an amphipathic channel with alternating hydrophobic clamps and electrostatic constrictions (SDUS geometry). PrankWeb pocket mapping and SwissDock simulations demonstrated that divalent cations (Ca²⁺, Mg²⁺), ATP and phosphorylated PHB–PHV oligomers occupy overlapping binding corridors, stabilized by aromatic, aliphatic and basic residues. Spectroscopic signatures of phosphate incorporation and aliphatic backbone structure corroborated in silico interaction models. This bifunctional architecture elevates PHAs from metabolic stores to active mediators of membrane homeostasis and stress adaptation. ORF1 defines a new class of polyesterbased channels, unifying biopolymer synthesis and transport within a single molecular scaffold, and offers a platform for engineering bespoke polymer conduits in synthetic biology. ORF1 polyhydroxyalkanoate polyester channel membrane topology AlphaFold2 PoreWalker molecular docking spectroscopy Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Polyhydroxyalkanoates (PHAs) represent a class of microbial polyesters that serve as dynamic carbon reserves and potential mediators of cellular homeostasis. While poly-(R)-3-hydroxybutyrate (PHB), the most ubiquitous PHA, has long been characterized as an energy storage granule in prokaryotes [ 1 , 2 ], emerging evidence suggests these biopolymers may play active roles in ion transport and stress response pathways [ 3 ]. This functional duality - as both metabolic deposit and putative signaling molecule - positions PHAs at the intriguing interface between microbial biochemistry and membrane biophysics. The ion transport hypothesis originated with Reusch and Sadoff's seminal demonstration that PHB-polyphosphate (polyP) complexes form Ca²⁺-selective channels in synthetic lipid bilayers [ 4 ]. Subsequent work extended this concept to eukaryotic systems, where PHB was implicated in modulating the mitochondrial permeability transition pore [ 5 ], though its precise role remains controversial [ 6 ]. Parallel studies established that synthetic PHB oligomers can self-assemble into ion-conductive structures [ 7 ], providing proof-of-concept for polyester-mediated transport. However, these abiotic systems lack the genetic programmability and regulatory precision of biological membranes. A critical unanswered question persists: do natural systems employ dedicated protein scaffolds to coordinate polyester biosynthesis and membrane transport? Canonical PHA synthases (PhaCs) are membrane-associated but lack transmembrane domains [ 8 ], while known ion channels show no polyester synthase activity. This functional divide suggests an uncharacterized class of proteins may exist at this biochemical intersection. Our discovery of a eukaryotic orf1 gene (HvORF1 from Hanseniaspora valbyensis ) that directs production of phosphorylated PHB-PHV copolymers in recombinant Escherichia coli [ 9 ] provides a potential missing link. Preliminary data suggest HvORF1 possesses both synthase activity and predicted transmembrane helices - a combination unprecedented in the literature. This study systematically evaluates whether HvORF1 represents the first known example of a genetically encoded, polyester-based transport system, potentially unifying decades of biochemical and biophysical observations. Results 1- Membrane Topology Predictions To investigate ORF1 membrane topology, we analyzed the full-length 344-residue amino acid sequence using six independent membrane topology prediction tools: MemBrain 3.1 [ 10 ], PolyPhobius [ 11 , 12 ], Philius [ 13 ], MEMSAT-SVM (Nugent & Jones, 2009), Phobius as described previously by Abd-El-Haleem et al. [ 9 ], and DeepTMHMM [14. All tools consistently predicted a central membrane-embedded domain, though the total number of predicted transmembrane helices (TMHs) varied. Initial Phobius analysis indicated four membrane-associated segments and suggested a potential pore-forming architecture, prompting further detailed analyses. The most extensive prediction came from MemBrain, identifying seven high-confidence transmembrane helices (TMH scores > 0.9) spanning residues T3–I34, L40–R68, E76–L110, F119–C148, L163–L200, A206-G235, and L242–S266, respectively. These helices alternate between cytoplasmic and extracellular loops, beginning with an extracellular N-terminus and ending with a cytoplasmic C-terminus (Fig. 1 A). Such multi-pass architecture, characterized by compact helical packing and alternating loops, resembles structural arrangements observed in secondary transporters [ 15 , 16 ] and mechanosensitive ion channels [ 17 , 18 ], supporting stable membrane integration and potential ion or substrate translocation functions. PolyPhobius, utilizing multiple sequence alignments and hidden Markov models, identified four transmembrane helices spanning residues 110–124, 126–140, 159–178, and 199–222. These helices formed an alternating topology (in–out–in–out), flanked by a cytoplasmic N-terminal region (residues 1–109) and a large cytoplasmic C-terminal tail (residues 223–344), defining a core membrane domain from residues 110 to 222 (Fig. 1 B). Despite predicting fewer helices than MemBrain, PolyPhobius captured nearly all core segments identified by other tools, emphasizing the conserved nature of this membrane-embedded region. MEMSAT-SVM (PSIPRED suite) predicted two TMHs spanning residues 163–178 (S1) and 200–215 (S2), and notably identified a re-entrant loop within residues 204–215 (Fig. 1 C). Re-entrant loops are critical structural motifs found in mechanosensitive and potassium channels, contributing to ion selectivity and gating mechanisms [ 18 , 19 ]. MEMSAT-SVM’s predicted topology, with an extracellular N-terminus and cytoplasmic C-terminus, aligns closely with orientations from MemBrain and PolyPhobius, strongly suggesting ORF1’s potential role in pore formation. Philius, employing dynamic Bayesian networks, identified a single major transmembrane helix (TMH) between residues 158–178, effectively partitioning the protein into a cytoplasmic N-terminal domain and an extracellular C-terminal domain (Fig. 1 D). Although simpler compared to MemBrain or PolyPhobius predictions, Philius independently confirmed membrane insertion within the core region consistently predicted by other tools. High-confidence scores strongly supported this membrane-spanning segment and indicated the absence of a signal peptide, suggesting direct co-translational membrane insertion. DeepTMHMM, leveraging transformer-based language modeling trained on curated membrane protein structural data, also predicted a single TMH spanning residues A207–A216, flanked by an intracellular N-terminal region (residues 1–206) and an extracellular C-terminal region (residues 217–344). This arrangement suggests a type I membrane protein topology, with the short, 10-residue TMH potentially serving as a membrane anchor, while the majority of functional domains reside in the cytoplasmic (N-terminal) and extracellular (C-terminal) compartments (Fig. 1 E). The predicted 10-residue transmembrane helix (TMH) is strikingly short compared to the ~ 20 residues typically required for an α-helical membrane span [ 20 ]. This unusual length suggests either significant structural flexibility or alternative membrane-association mechanisms, similar to pore-forming toxins like gramicidin that use β-strand architectures rather than α-helices for membrane integration [ 21 , 22 ]. Membrane Topology Consensus Mapping The remarkable convergence among predictions from Philius, DeepTMHMM, and MEMSAT-SVM on the same approximately 60-residue membrane-embedded region (residues 158–222) underscores the identification of a central structural domain potentially capable of ion transport or polymer translocation functions. To visualize the degree of overlap among independent tools, we constructed a comparative binary heatmap (Fig. 2 A) summarizing transmembrane predictions from five algorithms: MemBrain, PolyPhobius, MEMSAT-SVM, Philius, and DeepTMHMM. For each tool, the full-length ORF1 sequence (344 residues) was encoded as a binary vector where residues predicted to lie within transmembrane helices were assigned a value of 1, and all other residues (cytoplasmic, extracellular, or unassigned) were given a 0. These vectors were stacked across tools and visualized as a heatmap matrix, where the Y-axis corresponds to prediction tools and the X-axis corresponds to residue position. The resulting plot highlights a concentrated block of shared predictions within the 159–222 region, indicating broad inter-tool consensus in the absence of predefined thresholds or alignment constraints. To refine this consensus, we submitted ORF1 to TOPCONS2, a meta-predictor that integrates five topology algorithms. While the final consensus output did not annotate any transmembrane helices, three component tools—Philius, PolyPhobius, and SPOCTOPUS—independently predicted TM segments spanning residues 159–222 (Fig. 2 B). The TOPCONS2 reliability score began declining below 0.5 at residue 119, reached a minimum of 0.40 at residue 163, and rose sharply to 0.99 at residue 190, delineating a gradient typical of insertion transitions across the bilayer. This region also coincided with a ΔG of insertion minimum (~–1 kcal mol⁻¹), consistent with the thermodynamic profile of a membrane-embedded helix. The absence of this region from the final consensus reflects filtering effects due to disagreement from OCTOPUS and SCAMPI, rather than a lack of predictive evidence. Collectively, these findings define residues 159–222 as the most probable membrane-integrated domain of ORF1 (Fig. 2 C), supported by algorithmic convergence, biophysical favorability, and meta-consensus decomposition. 2- Pore Prediction To explore the functional implications of these topological predictions, the complete 344-residue ORF1 protein sequence was modeled using AlphaFold2 via the ColabFold pipeline, generating a three-dimensional structure in PDB format suitable for pore analysis. The predicted pore was oriented along the X-axis of the rotated structure and displayed a characteristic alternating geometry of cylindrical and conical segments, summarized by an SDUS pattern (Fig. 3 A). This geometry—consisting of a uniform entry (S), diverging cone (D), converging cone (U), and uniform exit (S)—is indicative of selective narrowing and widening critical for substrate gating. Diameter profiling at low resolution (3-Å intervals; Fig. 3 B) revealed substantial variations, with constrictions as narrow as 6.18 Å occurring around 13 Å along the pore axis, and prominent expansions reaching diameters of 17.31 Å (at ~ 10 Å) and 18.74 Å (at ~ 22 Å). These alternating constrictions and expansions suggest selective gating interspersed by broader internal cavities, characteristic of multiphase conduction channels. The analysis (1-Å intervals; Fig. 3 C) further delineated 47 cross-sectional slices spanning from approximately − 24.81 to + 24.18 Å along the pore axis. This detailed profiling highlighted narrow constrictions at the pore entrance (− 24 to − 18 Å) with minimum estimated diameters approaching ~ 1.0 Å. Occasional sub-angstrom values (~ 0.3 Å) were noted, but these likely represent artifacts due to local surface irregularities or computational resolution limits, as they were not consistently observed in the lower-resolution profile. A central segment (− 7 to + 15 Å) formed a plateau, maintaining diameters between 3.1 and 4.1 Å, followed by a maximal aperture of 6.99 Å near the pore exit. This asymmetric tapering strongly supports a unidirectional, selectively permeable conduit. Linearity analysis confirmed one dominant pore axis with an RMSD of 4.21 Å, exhibiting alternating straight (R) and curved (L) segments (Fig. 3 D). Approximately 10.5% of segments were perfectly linear, a hallmark of axial regularity necessary for efficient substrate transport. Residue-level analysis identified over 140 pore-lining amino acids, extending from MET1 (− 17.01 Å) to ARG344 (+ 21.96 Å). The N-terminal vestibule was enriched in hydrophilic (MET1, SER2, LEU27) and negatively charged residues (ASP72, SER73, GLU76), potentially acting as a selective cation entry region. Mid-pore segments displayed alternating hydrophobic (LEU89, ILE120, VAL123) and aromatic residues (TYR122, PHE128, TRP147), generating an amphipathic environment conducive to ion coordination and stabilization of hydrophobic regions of PHB–PHV copolymers. The distal cytoplasmic region, containing positively charged residues (ARG118, LYS117, ARG152), may form an electropositive exit zone, promoting directional substrate flow. Visualization of the pore geometry and cavity surface is shown in Fig. 3 E and F , with the protein oriented along the X-axis (lowest coordinate at the bottom). Red spheres mark pore centers at 1-Å intervals along the pore axis. Cross-sectional views illustrate pore architecture from different planes: XZ-plane sections for coordinates Y > 0 (top left) and Y 0 (bottom left) and Z < 0 (bottom right). Basic and hydrophobic residues predominate near the electrostatic pore exit, while polar residues line the dynamic vestibule region. This spatial arrangement supports ORF1’s potential function as an ion- or polymer-conducting channel. 1. Pocket Analysis P2Rank analysis of chain A in the ORF1 protein structure revealed eight distinct ligand-binding pockets (Fig. 4 ), each characterized by unique residue compositions and physicochemical profiles. These binding sites offer structural insight into the functional roles of ORF1 in ligand recognition, potential substrate gating, and polymer interactions, especially given its hypothesized involvement in phosphorylated PHB-PHV copolymer transport. Pocket 1 (score = 9.51, probability = 0.553) represents the highest-confidence binding site (Fig. 4 ). It comprises a network of hydrophobic and aromatic residues including Leu171 (0.7863), Leu174 (0.9894), Leu178 (0.4652), Lys180 (0.0812), Val185 (0.7794), Leu188 (0.3912), Phe189 (0.9011), and Tyr192 (1.2727), forming a deep polar cleft conducive to small hydrophobic ligand or polymer side-chain accommodation. Additional residues include Leu208 (0.4548), Met209 (1.1974), Gly212 (0.8103), Gly213 (0.5479), Ala216 (1.2716), Asn219 (0.0740), Ala220 (0.1847), Ser225 (0.2304), Phe227 (1.1972), Leu242 (1.0519), Phe244 (1.6216), Trp245 (0.6792), Phe246 (0.6837), Leu253 (1.5317), and Trp255 (1.0521). The presence of glycine, alanine, and serine residues suggests structural flexibility, potentially accommodating phosphate-linked moieties in the biopolymer. Hydrophobic clustering of leucines and phenylalanines may provide van der Waals stabilization similar to known polymer channels [ 23 ]. Pocket 2 (score = 8.06, probability = 0.473) overlaps partially with Pocket 1 and extends the binding environment through residues Leu163 (1.0496), Cys167 (0.9913), Ser170 (0.7953), Leu171, Leu174, Ile177 (0.3784), Leu178, Phe244, Phe246, Val249 (0.1314), Ile251 (0.8570), Leu253, Asp254 (0.5505), Trp255, Cys290 (0.5477), Met291 (1.8291), Val292 (0.9728), Val294 (0.6686), and Phe316 (0.5250). The inclusion of charged residues like Asp254 and polar aromatics (e.g., Phe316) suggests a mixed hydrophobic-hydrophilic tunnel; with electrostatic potential that may attract negatively charged phosphate groups. Pocket 3 (score = 6.99, probability = 0.396) is located at the N-terminal domain, comprising Met1 (1.0101), Phe24 (2.3542), Leu27 (2.6458), Val31 (0.2462), Leu40 (0.6937), Val41 (0.7409), Val42 (1.9468), Ser43 (1.5763), Ser45 (1.4089), Leu46 (2.2663), Pro47 (2.4572), and Leu55 (2.6646). This amphipathic surface, enriched in large hydrophobes and polar residues, resembles the entrance of amphipathic substrate channels, potentially accommodating hydrophobic polymer segments during insertion or export [ 24 ]. Pocket 4 (score = 2.23, probability = 0.054) includes Leu163, Phe164 (0.6762), Cys167, Phe189, Tyr192, Met209, and Trp255. These residues suggest a secondary aromatic-rich cavity capable of stabilizing π-interactions, possibly relevant for transient ligand stabilization during passage or anchoring (Robertson et al., 2005). Pocket 5 (score = 2.04, probability = 0.044) integrates Leu121 (1.2579), Leu296 (0.4532), Ser310 (1.1580), Val312 (0.4144), Val41, Ser43, Ile70 (0.4226), Asp72 (1.1802), Leu89 (0.6079), and Lys91 (1.5191). This cavity combines electrostatic potential from Asp72 and Lys91 with flexible loops, suggesting potential phosphate buffering capacity or transient ion coordination. Pocket 6 (score = 1.93, probability = 0.038) features Ser2 (0.0817), Leu13 (0.3785), Ser14 (0.0459), Asp308 (1.1620), Gly44 (1.3102), Ser45, Ser74 (0.5891), Asn93 (1.2309), and Lys95 (0.3267). The clustering of polar residues in this region supports its role as a hydrophilic interaction surface, potentially participating in pH or ion-induced gating changes [ 25 ]. Pocket 7 (score = 1.84, probability = 0.034) shares several residues with Pocket 6 and consists of Gly75 (0.5188), Glu76 (0.3200), Ala77 (0.4270), Ser45, Ser46, Ser74, and Lys95. This region may act as an auxiliary gate or substrate modulation surface based on solvent accessibility and side-chain polarity. Pocket 8 (score = 1.80, probability = 0.033) contains Phe164, Tyr168 (0.5425), Leu171, Val185, Trp186 (0.6912), Phe189, and Leu190 (0.1904). Although less probable, this pocket's hydrophobicity and high scoring residues suggest a lipid-facing concavity, consistent with anchoring or lateral diffusion functions observed in hydrophobic polymer channels. Several residues were shared among multiple predicted pockets, highlighting the interconnected nature of the ligand-accessible regions. For example, Leu171 and Leu174 appeared in Pockets 1, 2, and 8; Phe189 and Tyr192 were present in Pockets 1, 4, and 8; and both Trp255 and Phe244 were detected in Pockets 1 and 2. This pattern of shared residues across multiple pockets supports the presence of a structurally continuous tunnel-like feature rather than isolated cavities. Such overlapping hydrophobic and aromatic residues forming a common internal path are consistent with substrate-conducting tunnels described in pore-forming proteins, such as the mechanosensitive MscL channels [ 18 , 26 ] and bacterial porins [ 27 , 28 ]. Specifically, the recurrence of aromatic residues (e.g., Phe244, Trp255) suggests π-stacking capabilities and van der Waals complementarity that contribute to selective gating and stabilization of polymeric substrates. Similar architectures were observed in ABC transporters and PHB depolymerases, where tunnel-like conduits supported the unidirectional translocation of flexible, hydrophobic, or phosphorylated polymers [ 24 , 29 ]. These comparisons strongly support the hypothesis that ORF1 forms a membrane-associated conduit, and the recurring residues across Pockets 1–3 form the primary pathway for substrate passage. In contrast, Pockets 4–8 may represent side cavities or regulatory sites akin to allosteric modulation surfaces reported in modular transporters [ 30 , 31 ]. This residue-resolved pocket mapping, validated against the provided data and residue scores, offers mechanistic insights into the potential function of ORF1 in the transport or stabilization of phosphorylated PHB–PHV copolymers within engineered E. coli membranes. 4. Ligand Docking Validation SwissDock-based molecular docking was performed following pocket identification via PrankWeb (P2Rank), targeting the predicted channel lumen of the AlphaFold2-modeled ORF1 protein. The primary objective was to validate whether the predicted transmembrane pore serves as a selective conduit for charged ligands and extended phosphorylated polymers, with docking simulations performed for Ca²⁺, Mg²⁺, ATP, and a model of a phosphorylated PHB–PHV co-polymer. The docking of Ca²⁺ and Mg²⁺ ions (Fig. 5 A-B), modeled as spherical species with + 2 charges, confirmed the presence of electronegative coordination zones within PrankWeb Pocket 2 (score = 8.06, probability = 0.473) and Pocket 4 (score = 2.23, probability = 0.054). Acidic residues ASP72 (score: 1.1802), ASP254 (0.5505), ASP286 (0.0760), ASP289 (0.5582), and GLU88 (0.3069) served as key coordinating anchors. These residues, localized on extracellular loops or helices, formed canonical carboxylate-rich ion-binding sites consistent with cation filters observed in channels such as CorA and Orai1 [ 32 , 33 ]. Surrounding polar residues including ASN93 (1.2309), SER73 (0.4708), and SER310 (1.1580) enhanced coordination geometry by hydrogen bonding, indicating a vestibular capture-and-release mechanism for divalent cations, particularly near the entrance of the predicted pore pathway outlined by PoreWalker. The phosphorylated co-polymer was docked using a model based on a repeating unit with the SMILES: CC(C)COC(= O)CCOP(= O)(O)OCC(C)CCOC(= O)CC , representing the bifunctional structure of PHB-P-PHV (Fig. 5 C-D). The resulting poses occupied the full length of the predicted channel axis. This alignment overlapped with PrankWeb Pocket 1 (score = 9.51, probability = 0.553) and Pocket 2, implicating residues such as LEU13 (0.3785), ILE34 (0.2330), LEU174 (0.9894), LEU178 (0.4652), VAL249 (0.1314), and LEU253 (1.5317) in backbone hydrophobic anchoring. These residues, mostly from helix-facing surfaces, create a continuous aliphatic corridor mimicking lipid bilayer interiors [ 34 ]. Furthermore, aromatic residues PHE244 (1.6216), TRP245 (0.6792), and TYR192 (1.2727) formed mid-channel π-stacking zones and CH–π interactions stabilizing polymer side chains—a mechanism documented in bacterial transporters for large hydrophobic molecules [ 35 , 36 ]. ARG311 and ARG317 provided terminal salt-bridge anchoring of the copolymer’s phosphate moiety, forming guanidinium–phosphate interactions also seen in ATPases and anion channels [37. CYS159 (1.1619) and CYS290 (0.5477) were positioned to participate in thiol–phosphate or redox-mediated interactions, possibly modulating dynamic channel gating via disulfide formation. ATP (SMILES: Nc1ncnc2c1ncn2[C@H]1O[C@@H](COP(= O)(O)OP(= O)(O)OP(= O)(O)O)[C@H](O)[C@H]1O) was docked to investigate nucleotide-coupled activity. Binding localized at the cytoplasmic side (Fig. 5 E-F), consistent with Pocket 2 and proximal to PrankWeb Pocket 6 (score = 1.93, probability = 0.038). Positively charged residues ARG68 (0.0835), LYS91 (1.5191), LYS95 (0.3267), and ARG311 were involved in anchoring the triphosphate chain via electrostatic interactions, while PHE189 (0.9011) and PHE316 (0.5250) formed a sandwich-like binding cleft around the adenine base—echoing canonical ATP-binding folds found in ABC transporter NBDs [ 29 ]. Hydroxyl moieties of the ribose interacted with SER170 (0.7953) and SER310 (1.1580), providing fine-tuned specificity. CYS167 and GLU88, found near the ligand cavity, potentially modulate ATP binding through local redox status or microenvironmental pKa variations, paralleling control mechanisms reported in cysteine-based regulatory channels [ 38 ]. Residues such as ARG311, SER310, PHE316, and CYS290 appeared in multiple ligand interactions (ATP, PHB–PHV, and ions), signifying their multifunctional role in channel activity. These residues clustered near the central axis of the PoreWalker-predicted lumen, supporting their function as selectivity and gating determinants. PrankWeb results confirmed these residues to be part of the highest-scoring pockets (Pockets 1 and 2), reinforcing the hypothesis of a continuous binding groove. Moreover, the frequent reuse of residues—LEU171, LEU174, PHE189, TYR192, and TRP255—across docking scenarios and their consistent appearance in overlapping P2Rank pockets further supports the notion of an integrated translocation tunnel. This architectural convergence mirrors structural models of selective channels in lipid-polymer conjugate systems [ 19 , 39 , 40 ]. Spectroscopic corroboration of the ORF1PHB-P-PHV copolymer interaction model Fouriertransform infrared (FTIR) spectroscopy of the biosynthesized PHBPPHV copolymer [ 9 ] displays a diagnostic set of seventeen bands that align precisely with the chemical features required for docking into the ORF1 pore. Prominent P–O and P–O–C vibrations at 1,207, 1,076, 833 and 542 cm⁻¹ confirm covalent phosphate incorporation; this anionic handle is the same motif that SwissDock predicts to form phosphate salt bridges with Arg311 and Arg317 at the channel’s constriction point. Intense aliphatic C–H stretches (2,928 and 2,870 cm⁻¹) and bending modes (1,450 and 1,367 cm⁻¹) attest to a CHrich backbone, matching the Leu/Ile/Vallined hydrophobic tunnel defined by membranetopology and PoreWalker analyses. A single ester C = O band at 1,724 cm⁻¹ confirms that each 3hydroxybutyrate/3hydroxyvalerate unit is neutral and esterlinked, consistent with the dipole–π contacts observed in docking between the polymer carbonyls and aromatic pore residues (Phe244, Tyr192, Trp255). Finally, highfrequency O–H stretches at 3,782, 3,697 and 3,173 cm⁻¹ indicate residual terminal hydroxyls that rationalize the hydrogenbond network predicted between the polymer’s leading edge and Ser73, Ser170 and Ser310 at the vestibule and midpore. ¹H NMR spectroscopy of the phosphorylated PHB–PHV copolymer reinforces the channelpolymer interaction model derived from topology and docking mapping. Upfield signals at 0.87 ppm (terminal CH₃) and 1.25–1.32 ppm (internal CH₂) attest to a long, flexible aliphatic backbone that complements the Leu/Ile/Vallined hydrophobic tunnel of Pocket 1, while the branched 3hydroxyvalerate methyl resonance at 1.60 ppm accounts for steric packing against Leu174 and Val249. Carbonyladjacent methylenes at 2.16 and 2.38 ppm confirm ester linkages whose dipoles engage in the πstacking niche formed by Phe244, Tyr192 and Trp255, and the distinctive 2.48 ppm CH₂–O–P signal provides direct evidence of phosphate grafting that SwissDock predicts to anchor via Arg311 and Arg317. Midfield resonances at 3.17, 4.02 and 4.14 ppm arise from CH₂–O–C(= O) and CH₂–O–P groups capable of hydrogenbonding to Ser73, Ser170 and Ser310 at the vestibule and midpore, whereas the 3.29 ppm terminal CH₂–OH peak rationalises the hydroxylmediated ‘handshake’ that initiates polymer entry. A backbone methine at 3.65 ppm and a deshielded O–CH–C(= O) methine at 5.22 ppm indicate chiral, esteradjacent protons consistent with the elongated, partially ordered pose accommodated within the tapered lumen detected by PoreWalker. ¹³C NMR analysis of the phosphorylated PHB–PHV copolymer further substantiates the structural complementarity between polymer chemistry and the ORF1 channel. A cluster of aliphatic signals at 14.2 ppm (terminal CH₃) and 22.8–30.3 ppm (internal CH₂) confirms a long hydrocarbon backbone that matches the Leu/Ile/Val hydrophobic corridor delineated by topology prediction and SwissDock. The unique 32.0 ppm resonance, assigned to CH₂ directly bonded to phosphate (–CH₂–O–P), provides carbonlevel evidence of covalent phosphorylation and supports the phosphate salt bridges predicted for Arg311 and Arg317 at the pore constriction. Three oxygenbearing methylenes at 76.9–77.4 ppm (–CH₂–O–) indicate multiple ester/ether linkages capable of hydrogenbonding to Ser73, Ser170 and Ser310 along the channel wall. Finally, two sharp carbonyl peaks at 173.7 and 178.5 ppm confirm fully esterified 3hydroxybutyrate/3hydroxyvalerate units, whose dipoles and πfaces engage the aromatic midpore niche formed by Phe244, Tyr192 and Trp255 in docking simulations. Electronimpact GC–MS of the methanolysed PHBPPHV product yields a progressive series of phosphate and estercontaining fragments that dovetail with the SwissDock residueinteraction map. Lowmass ions at m/z 45 (C₂H₅O⁺) and 73 (trimethylsilyl, CH₃O₃⁺) verify simple hydroxyalkanoate units and oxygenated endgroups characteristic of PHA backbones. Successive phosphatebearing fragments— m/z 207 (C₆H₇OP⁺) and 267 (C₈H₁₁OP⁺)—demonstrate that phosphate is covalently attached to short alkyl segments rather than present as free polyphosphate, matching the singlephosphate anchor required for saltbridge formation with Arg311 and Arg317 at the ORF1 constriction. Highermass ions at m/z 325 (C₁₀H₁₃O₈P⁺) and 341 (C₁₁H₁₅O₈P⁺) contain both multiple ester linkages and a single phosphate, mirroring the mixed polar/hydrophobic surface that docks against the Ser/Thr hydrogenbond network and the Leu/Ile/Val belt along the channel wall. The dominant highmass fragment at m/z 429 (C₁₅H₂₅O₉P₃⁺) confirms the presence of extended alkyl chains bearing three phosphate groups, consistent with the multiphosphate docking poses that span Pocket 1 and Pocket 2 and engage the recurrent basic cluster (Arg68, Lys91, Lys95, Arg311). Collectively, the phosphatetoalkyl progression from m/z 207 to 429 provides compositional evidence for a polymer that is simultaneously hydrophobic and highly phosphorylated—the precise duality required for stable, geometrymatched threading through the amphipathic ORF1 pore predicted by topology, PoreWalker and SwissDock analyses. In the FTIR set, 4 of 17 bands (23%) are phosphate-specific—comprising one P = O stretch and three P–O or P–O–C vibrations—while only a single ester carbonyl peak is observed, yielding a phosphate-to-carbonyl peak ratio of 4:1. This unusually high phosphate signature for polyhydroxyalkanoates suggests strong interaction with basic residues such as Arg and Lys, particularly in the high-probability binding pockets identified in the PrankWeb analysis (Pocket 1 and Pocket 2). By contrast, the ¹H NMR spectrum includes only one CH₂–O–P resonance among 14 non-solvent signals (7%), and the ¹³C NMR data similarly shows one phosphate-bound carbon resonance out of 12 peaks (8%), indicating that phosphate incorporation is spatially localized rather than uniformly distributed along the polymer chain. This pattern aligns with SwissDock results where discrete phosphate “nodes” form electrostatic contacts with ARG311 and ARG317, while intervening aliphatic segments transit the hydrophobic tunnel. Supporting this localization hypothesis, GC–MS analysis reveals that five out of nine prominent fragment ions (56%) retain phosphorus (m/z 207, 267, 325, 341, 429), indicating enhanced mechanical stability of phosphate-containing regions during fragmentation. Such resilience is expected if phosphate sites serve as anchoring clamps, consistent with directional threading through a transmembrane channel. Collectively, the increasing phosphate signature from NMR (≈ 8%) to FTIR (23%) to GC–MS (56%) mirrors the predicted movement of the polymer through the ORF1 pore: sparse phosphate termini initiate capture at the vestibule, mid-pore anchoring occurs at the basic clamp, and polyphosphate-rich segments emerge at the cytosolic face. These findings converge with the structural and computational model illustrated in Fig. 6 , confirming that the PHB–P–PHV copolymer engages the ORF1 protein in a manner consistent with ion-channel-like unidirectional translocation. Discussion Our findings position ORF1 as the first genetically encoded polyester synthase–translocator, expanding the paradigm established by membraneintegrated glycosyltransferases. In cellulose synthase, the GTA fold of BcsA is coupled to a helical transmembrane pore in BcsB, enabling simultaneous polymer elongation and extrusion [ 41 , 42 ]. ORF1 similarly merges a catalytic PHA synthase domain with a compact, multihelix core (residues 159–222), suggesting an evolutionary convergence on bifunctional polymerase–channel architectures. Unlike polysaccharide channels, however, ORF1 exploits polyester chemistry—alternating hydrophobic and phosphatebearing moieties—to regulate its gating and substrate specificity. Beyond carbon storage, PHAs serve as dynamic mediators of microbial stress adaptation. Psychrophilic Arctic isolates exhibit upregulated PHB granule mobilization under freeze–thaw cycles, where polymer–membrane interactions stabilize lipid phases [ 43 ]; similarly, halophiles deploy PHA synthesis to buffer osmotic shock, linking polyester turnover to ion homeostasis [ 44 ]. ORF1’s ability to manufacture and channel phosphorylated PHB–PHV copolymers thus provides a mechanistic basis for rapid, localized PHA deployment at the membrane, coupling energy mobilization with stressresponsive ion flux. Mechanosensitive channels like MscS exemplify how compact helical bundles and reentrant loops coordinate gating under tension. Highresolution structures of EcMscS reveal a sevenhelix transmembrane assembly that dilates via hingelike rearrangements in response to bilayer stress [ 45 ]; functional studies implicate peripheral arginines as electrostatic clamps that stabilize open and closed states [ 17 ]. ORF1 similarly positions basic residues (Arg311, Arg317) at constriction points and interdigitates hydrophobic helices to create alternating constrictions and vestibules. We propose that polymer length and degree of phosphorylation modulate pore tension in a manner analogous to pressureinduced gating of MscS, but rectified by multivalent electrostatic “clamps” on the polymer backbone. Our pocket mapping and docking analyses further reveal a continuous, amphipathic tunnel reminiscent of ABC importers. In the vitamin B₁₂ transporter BtuCD, substrate passage occurs through an alternatingaccess pore gated by ATPdriven conformational cycles [ 29 ]. ORF1 diverges by eliminating nucleotidebinding domains, instead harnessing polymer binding energy and local electrostatics to ratchet polyester movement. Shared aromatic and aliphatic residues create πstacking niches and van der Waals corridors that likely stabilize transient binding of the polyester chain, while charged residues serve as directional anchors. This mechanism parallels the Brownian ratchet models described for protein translocation, repurposed here for polyester substrates. Our integrative spectroscopic–computational validation underscores the complementarity between polymer chemistry and channel architecture. FTIR bands diagnostic of phosphate incorporation (e.g., 1,207 and 1,076 cm⁻¹) align with predicted saltbridge interactions at Arg311/Arg317, while NMR resonances pinpoint polymer moieties that engage hydrophobic belts and electrostatic vestibules. Similar correlative approaches in cellulose synthase studies have linked in vitro spectroscopy with pore occlusion models, reinforcing the value of combined biophysical and structural methods [ 41 ]. By triangulating topology predictions, docking profiles, and spectral fingerprints, we provide a cohesive model of polyester threading that transcends individual techniques. Conclusion We have demonstrated that ORF1 from Hanseniaspora valbyensis constitutes the first example of a genetically encoded polyester synthase–translocator, uniquely coupling PHA polymerization with vectorial export of phosphorylated PHB–PHV copolymers through a structurally defined amphipathic pore. Integrative membrane topology predictions, AlphaFold2 modeling, and PoreWalker analyses unveil a central helical core (residues 159–222) that orchestrates alternating hydrophobic clamps and electrostatic gates, while docking and spectroscopic data confirm the molecular interactions driving polymer threading. This dualfunction architecture elevates PHAs from inert carbon stores to active participants in membrane homeostasis and stress response, revealing an evolutionary strategy for integrating metabolic reserve mobilization with ion and polymerconductive behavior. ORF1 thus expands the functional repertoire of microbial polyesters and uncovers a paradigm for polyesterbased channels that parallels glycosyltransferase and ABC transporter mechanisms but exploits unique polyester chemistry. Future efforts should focus on reconstitution of ORF1 in defined lipid systems, singlechannel electrophysiology, and cryoEM of polymerbound states to resolve gating dynamics and validate conductance. Genome mining across eukaryotes will ascertain the prevalence of synthase–translocator homologs, while protein engineering may harness ORF1’s bifunctional scaffold to create bespoke polymer channels for biotechnological and syntheticbiology applications. By uniting biopolymer synthesis and membrane translocation within a single molecular machine, ORF1 opens a new frontier in our understanding and exploitation of microbial polyester biochemistry. Methods The Hanseniaspora valbyensis ORF1 gene (1,035 nucleotides) was cloned into pET-28a(+) and expressed in Escherichia coli BL21(DE3) Δ phaC for heterologous production of phosphorylated PHB–PHV, as previously described [ 9 ]. Phosphorylated PHB–PHV was biosynthesized, purified, and validated by transmission electron microscopy (TEM), Fourier-transform infrared spectroscopy (FTIR), nuclear magnetic resonance (NMR), and gas chromatography–mass spectrometry (GC–MS). The ORF1 coding sequence was identified using NCBI ORFfinder https://www.ncbi.nlm.nih.gov/orffinder with default parameters (ATG start codon, minimum length 300 nucleotides), excluding nested ORFs; translation yielded a 344-residue protein ( sequences provided in Supplementary Data 1 ). To determine the membrane topology of ORF1, six complementary algorithms were applied to the full-length 344-residue amino acid sequence. Phobius [described at 9] and PolyPhobius ( http://phobius.sbc.su.se/ ) , both employing hidden Markov models—with the latter incorporating multiple sequence alignments—were used to identify potential transmembrane helices and signal peptides. MEMSAT-SVM, accessed via the PSIPRED server ( https://bioinf.cs.ucl.ac.uk/psipred/ ) , utilized support vector machines trained on structural datasets to predict membrane-spanning helices and re-entrant loops. Philius ( http://www.yeastrc.org/philius ) leveraged dynamic Bayesian networks to infer transmembrane topologies. DeepTMHMM ( https://dtu.biolib.com/DeepTMHMM ) , a state-of-the-art neural transformer-based model, was employed to refine transmembrane boundaries with sub-residue precision. In parallel, MemBrain 3.1 ( http://www.csbio.sjtu.edu.cn/bioinf/MemBrain/ ) integrated statistical learning and residue physicochemical properties to define helical regions with membrane insertion potential. To assess membrane topology consensus for the ORF1 protein, we utilized TOPCONS2 ( https://topcons.net/pred/ ), a widely adopted meta-prediction server that integrates five independent topology algorithms: OCTOPUS, Philius, PolyPhobius, SCAMPI, and SPOCTOPUS. The full-length ORF1 amino acid sequence (344 residues) was submitted through the default pipeline using the TOPCONS2 web interface. The platform outputs per-residue topology assignments (cytoplasmic, transmembrane, extracellular), transmembrane helix orientation (IN→OUT or OUT→IN), and signal peptide predictions. TOPCONS2 also provides two quantitative profiles: (i) a per-residue reliability score, derived from the agreement among predictors, and (ii) a ΔG insertion energy profile, estimating the free energy cost (in kcal mol⁻¹) of inserting each residue into the membrane based on an experimentally derived biological hydrophobicity scale. Graphical outputs include individual prediction tracks for each integrated algorithm, a consensus topology map, and two continuous plots for the reliability and ΔG insertion profiles. For structural modeling, AlphaFold2 was executed via the ColabFold platform ( https://colabfold.mmseqs.com ) , using MMseqs2-based homology detection and AlphaFold’s Evoformer network to generate high-accuracy three-dimensional structures. To characterize potential pore formation, the AlphaFold2 model was submitted to PoreWalker ( https://www.ebi.ac.uk/thornton-srv/software/PoreWalker/ ) , a cavity and channel analysis algorithm that identifies transmembrane pores, computes diameter profiles at 1 Å resolution, classifies geometric segments (SDUS: Straight–Diverging–Uniform–Straight), and maps pore-lining residues. Ligand-binding site predictions were performed using Prank2 (PrankWeb, https://prankweb.cz/ ), a machine learning-based platform developed by Charles University as part of ELIXIR's European bioinformatics infrastructure. The AlphaFold2 structure of ORF1 was analyzed using the platform's default parameters with the original structure configuration. The algorithm identified and ranked potential binding pockets based on volume, surface area, and druggability scores. Protein structure visualization and cavity property mapping were conducted using the integrated PrankWeb visualization tools. Molecular docking simulations were performed using SwissDock ( http://www.swissdock.ch ) , where the AlphaFold2 structure of ORF1 was used as a rigid receptor. Ligands included divalent cations Ca²⁺ and Mg²⁺ (modeled as charged spheres), ATP (structure retrieved from PubChem and optimized using Open Babel and Avogadro), and a biosynthetic oligomeric PHB–PHV–phosphate construct, reflecting the spectroscopically characterized polymer [ 9 ]. Binding poses were ranked based on full fitness scores and ΔG binding affinity. Molecular interactions and ligand placements were visualized using UCSF ChimeraX ( https://www.cgl.ucsf.edu/chimerax/ ). Spectroscopic validation of the polymer’s phosphate and ester functionalities was derived from previously reported FTIR, ¹H NMR, and ¹³C NMR signals [ 9 ] consistent with phosphorylated co-polyesters. GC–MS data supported the presence of mono-, di-, and triphosphorylated oligomers aligning with ligand docking models. Phosphatetocarbonyl ratios in spectroscopic data were calculated as band or peak count divided by total bands. Two-dimensional data visualizations were generated using Matplotlib 3.8 in Python 3.11. All graphical outputs maintained the native resolution and quality parameters of the respective software tools. Declarations Data availability: All data generated or analyzed during this study are included in this published article. Ethics approval and consent to participate : Not applicable. Consent for publication : The author has read and approved the manuscript and consent to its publication. Competing interests : The author declares no competing interests. Funding : This research received no external funding. Clinical trial: no clinical trial registration is applicable Author contributions : Dr. Desouky Abd‑El‑Haleem conceptualized, developing the methodological framework encompassing membrane‑topology prediction, structural modeling, pore analysis and molecular docking. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6506981","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":455622261,"identity":"e03a776f-456f-4328-9e3b-fc3ef2d715a8","order_by":0,"name":"Desouky A.M. Abd-El-Haleem","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA9ElEQVRIiWNgGAWjYDACCQYDhocNDDJgzgcGhgSIICEtiQ0MPCA24wyStTDzEKOFf3bzxgeJO+x4+Nm7Uzfbttnl8bM3MN74gM+SO8eKDRLPJPNI9pzddju3LblYsucAs+UMfNbcyDGTSGxj5jG4kQvSwpy44UYCmzQPHh3yN3LMfyS21fPY33+77bZlWz1Eyx88WgyAtjAkth3mMZDg3Xabse0wRAs+dxkC/QJ02HEeiTO52272nDueOLPnYLNlDx4tcrebN3742FYtx99+dtuNH2XVif3szQdv/MBnDQpgZAOTDURrAAJ83h4Fo2AUjIIRCwBHxlZM6ni8/QAAAABJRU5ErkJggg==","orcid":"","institution":"City of Scientific Research and Technological Applications","correspondingAuthor":true,"prefix":"","firstName":"Desouky","middleName":"A.M.","lastName":"Abd-El-Haleem","suffix":""}],"badges":[],"createdAt":"2025-04-22 19:38:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6506981/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6506981/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":82656181,"identity":"44dd0dbd-2386-476b-bf9c-481f79428f81","added_by":"auto","created_at":"2025-05-13 18:51:25","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1041921,"visible":true,"origin":"","legend":"\u003cp\u003eMembrane topology predictions of the ORF1 protein from \u003cem\u003eHanseniaspora valbyensis\u003c/em\u003e. Predictions from (\u003cstrong\u003eA\u003c/strong\u003e) MemBrain, (\u003cstrong\u003eB\u003c/strong\u003e) PolyPhobius, (\u003cstrong\u003eC\u003c/strong\u003e) MEMSAT-SVM, (\u003cstrong\u003eD\u003c/strong\u003e) Philius, and (\u003cstrong\u003eE\u003c/strong\u003e) DeepTMHMM\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-6506981/v1/98fa2553478a0996c8a3291b.png"},{"id":82655851,"identity":"a102ac93-7f91-4b96-a5a0-baaf336933a3","added_by":"auto","created_at":"2025-05-13 18:43:25","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":567811,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eConsensus prediction of transmembrane regions in ORF1. (A)\u003c/strong\u003eComparative heatmap showing binary transmembrane predictions across five topology tools (MemBrain, PolyPhobius, MEMSAT-SVM, Philius, and DeepTMHMM) for each residue in the 344-amino-acid ORF1 protein. Yellow blocks indicate predicted transmembrane helices (TMHs), with a convergence zone spanning residues 159–222 identified by ≥3 predictors. \u003cstrong\u003e(B)\u003c/strong\u003e TOPCONS2 output integrating five algorithms. Grey and white bars represent TMHs with IN→OUT and OUT→IN orientations, respectively; red/blue lines indicate cytoplasmic/extracellular sides.\u003cbr\u003e\n \u003cstrong\u003e(C)\u003c/strong\u003eTOPCONS reliability profile across the ORF1 sequence. A sharp dip in reliability (to 0.40 at residue 163) followed by recovery (to ~0.99 by residue 190) further delineates the 159–222 segment as a putative membrane-embedded domain.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-6506981/v1/d665f3a9acba336dbf0a74a8.png"},{"id":82656182,"identity":"18eb2bac-1d03-4537-8e49-c74f8229730e","added_by":"auto","created_at":"2025-05-13 18:51:25","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2079192,"visible":true,"origin":"","legend":"\u003cp\u003eStructural modeling and pore analysis of the ORF1 protein using AlphaFold2 and PoreWalker. (\u003cstrong\u003eA\u003c/strong\u003e) Simplified schematic illustrating the SDUS (uniform entry [S], diverging cone [D], converging cone [U], uniform exit [S]) geometry of the pore. (\u003cstrong\u003eB\u003c/strong\u003e) Lower-resolution (3 Å intervals) diameter profile along the full pore length, emphasizing gating points and cavity expansions typical of multiphase conduction channels. \u003cstrong\u003e(C)\u003c/strong\u003e High-resolution (1 Å intervals) diameter profile showing pore diameters (Dx) across cross-sectional slices along the pore axis (X-coordinate). (\u003cstrong\u003eD\u003c/strong\u003e) Linear representation of pore axis straightness, with segments alternating between straight (R) and curved (L) patterns; RMSD and straightness metrics shown. (\u003cstrong\u003eE\u003c/strong\u003e) Surface representation of the predicted pore (green), with the central pore axis marked by red pseudo-atoms spaced at 3 Å intervals. (\u003cstrong\u003eF\u003c/strong\u003e) Cross-sectional surface depiction highlighting pore-lining residues and central axis markers.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6506981/v1/0838a2f4199a3030b0a9ecfe.png"},{"id":82655856,"identity":"7a3dbb11-69f7-4c78-90e8-d38bc07c2931","added_by":"auto","created_at":"2025-05-13 18:43:25","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2429043,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePredicted ligand-binding pockets in ORF1.\u003c/strong\u003e Surface representation of the full-length ORF1 structure (left) with all eight P2Rank-predicted pockets (P1–P8). Colour coding: P1, red; P2, yellow; P3, orange; P4, light blue; P5, green; P6, dark blue; P7, pink; P8, magenta. Grey ribbon, protein backbone. \u003cstrong\u003e(b–i)\u003c/strong\u003e Close-up views of individual pockets (same colour scheme) showing side-chain sticks of every residue assigned by P2Rank. P1 forms an elongated, hydrophobic corridor spanning two helices; P2 occupies an adjacent cleft that partially overlaps with P1; P3 resides in the N-terminal amphipathic surface; P4–P8 are smaller cavities located on lateral or cytoplasmic faces\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-6506981/v1/fc45ac9772333d463dc9d350.png"},{"id":82656189,"identity":"2d43c198-5d0d-4ab0-81ab-0bfb8d44dc19","added_by":"auto","created_at":"2025-05-13 18:51:26","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":3302380,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMolecular docking of ions and ligands onto the predicted AlphaFold2 ORF1 structure using SwissDock and visualized in ChimeraX.\u003c/strong\u003e \u003cstrong\u003e(A, B)\u003c/strong\u003e Divalent calcium (orange spheres) and magnesium (green spheres) ions docked onto the ORF1 structure, visualized in ribbon \u003cstrong\u003e(A)\u003c/strong\u003e and surface \u003cstrong\u003e(B)\u003c/strong\u003erepresentations. \u003cstrong\u003e(C)\u003c/strong\u003e Atomic models of Ca²⁺ and Mg²⁺ used in docking. \u003cstrong\u003e(D, E)\u003c/strong\u003e Binding of the phosphorylated PHA copolymer (PHB–P–PHV) docked into a structured cleft formed by β-sheets and surrounding helices. The ligand is shown in stick representation, with carbon (beige), oxygen (red) and phosphorus (orange) atoms highlighted. \u003cstrong\u003e(F)\u003c/strong\u003e Structural representation of the PHB–P–PHV ligand. \u003cstrong\u003e(G, H)\u003c/strong\u003e ATP binding to the same cavity reveals a defined interaction site, distinct from the PHA-binding region. Ribbon \u003cstrong\u003e(G)\u003c/strong\u003e and surface \u003cstrong\u003e(H)\u003c/strong\u003e views illustrate ATP interactions localized near the central β-sheet scaffold. \u003cstrong\u003e(I)\u003c/strong\u003e The ATP structure used in docking\u003c/p\u003e","description":"","filename":"Figure51.png","url":"https://assets-eu.researchsquare.com/files/rs-6506981/v1/34bdd68a7a1e990973c1f025.png"},{"id":82656185,"identity":"bd2ec4ee-cddf-4104-b56a-f98fa2d11e70","added_by":"auto","created_at":"2025-05-13 18:51:25","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":902428,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMultimodal structural and spectroscopic validation of a directional ion-polymer channel formed by ORF1 and PHB–P–PHV copolymer.\u003c/strong\u003e \u003cstrong\u003e(A)\u003c/strong\u003eOverlay of FTIR and ¹H NMR spectra showing key vibrational and proton signals associated with ester carbonyl (~1724 cm⁻¹, ~2.48 ppm) and phosphate moieties (~1076 and 1207 cm⁻¹), supporting the presence of phosphate-linked aliphatic bridges. \u003cstrong\u003e(B) \u003c/strong\u003eAnnotated spectral motif of the polymer, indicating ester linkages, CH₂–O–P phosphate nodes, and C=O carbonyl groups from polyhydroxyalkanoate units; labels match assignments confirmed in FTIR, ¹H, and ¹³C NMR data. \u003cstrong\u003e(C)\u003c/strong\u003eSchematic representation of docking results showing the alignment of the PHB–P–PHV copolymer, ATP, and divalent cations (Mg²⁺ and Ca²⁺) within the ORF1 pore, highlighting polymer threading through a unidirectional tunnel formed by hydrophobic walls and charged gates. \u003cstrong\u003e(D\u003c/strong\u003e) Zoomed view of the mid-pore region where ester groups (C=O at ~173 ppm in ¹³C NMR) form stabilizing interactions with the pore wall, constituting a mid-pore ester clamp for polymer anchoring.\u003cbr\u003e\n \u003cstrong\u003e(E\u003c/strong\u003e) Illustration of directional extrusion model, in which phosphate-rich polymer is guided through the ORF1 channel via hydrophobic and electrostatic interactions, terminating in a basic residue gate at the cytoplasmic side\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-6506981/v1/60c2d3467fd001e8fe6c636b.png"},{"id":84774360,"identity":"60ad61e8-5f54-438d-b6b9-08fda5b4bd3f","added_by":"auto","created_at":"2025-06-17 08:40:02","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":14889994,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6506981/v1/491f3a9c-482a-4779-9343-6ca16fa6a261.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Bifunctional Polyester Synthase–Channel Driving Phosphorylated PHB–PHV Synthesis and Ion Conductance","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePolyhydroxyalkanoates (PHAs) represent a class of microbial polyesters that serve as dynamic carbon reserves and potential mediators of cellular homeostasis. While poly-(R)-3-hydroxybutyrate (PHB), the most ubiquitous PHA, has long been characterized as an energy storage granule in prokaryotes [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], emerging evidence suggests these biopolymers may play active roles in ion transport and stress response pathways [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. This functional duality - as both metabolic deposit and putative signaling molecule - positions PHAs at the intriguing interface between microbial biochemistry and membrane biophysics.\u003c/p\u003e \u003cp\u003eThe ion transport hypothesis originated with Reusch and Sadoff's seminal demonstration that PHB-polyphosphate (polyP) complexes form Ca\u0026sup2;⁺-selective channels in synthetic lipid bilayers [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Subsequent work extended this concept to eukaryotic systems, where PHB was implicated in modulating the mitochondrial permeability transition pore [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], though its precise role remains controversial [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Parallel studies established that synthetic PHB oligomers can self-assemble into ion-conductive structures [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], providing proof-of-concept for polyester-mediated transport. However, these abiotic systems lack the genetic programmability and regulatory precision of biological membranes.\u003c/p\u003e \u003cp\u003eA critical unanswered question persists: do natural systems employ dedicated protein scaffolds to coordinate polyester biosynthesis and membrane transport? Canonical PHA synthases (PhaCs) are membrane-associated but lack transmembrane domains [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], while known ion channels show no polyester synthase activity. This functional divide suggests an uncharacterized class of proteins may exist at this biochemical intersection.\u003c/p\u003e \u003cp\u003eOur discovery of a eukaryotic orf1 gene (HvORF1 from \u003cem\u003eHanseniaspora valbyensis\u003c/em\u003e) that directs production of phosphorylated PHB-PHV copolymers in recombinant \u003cem\u003eEscherichia coli\u003c/em\u003e [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] provides a potential missing link. Preliminary data suggest HvORF1 possesses both synthase activity and predicted transmembrane helices - a combination unprecedented in the literature. This study systematically evaluates whether HvORF1 represents the first known example of a genetically encoded, polyester-based transport system, potentially unifying decades of biochemical and biophysical observations.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e1- Membrane Topology Predictions\u003c/h2\u003e \u003cp\u003eTo investigate ORF1 membrane topology, we analyzed the full-length 344-residue amino acid sequence using six independent membrane topology prediction tools: MemBrain 3.1 [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], PolyPhobius [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], Philius [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], MEMSAT-SVM (Nugent \u0026amp; Jones, 2009), Phobius as described previously by Abd-El-Haleem et al. [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], and DeepTMHMM [14. All tools consistently predicted a central membrane-embedded domain, though the total number of predicted transmembrane helices (TMHs) varied. Initial Phobius analysis indicated four membrane-associated segments and suggested a potential pore-forming architecture, prompting further detailed analyses. The most extensive prediction came from MemBrain, identifying seven high-confidence transmembrane helices (TMH scores\u0026thinsp;\u0026gt;\u0026thinsp;0.9) spanning residues T3\u0026ndash;I34, L40\u0026ndash;R68, E76\u0026ndash;L110, F119\u0026ndash;C148, L163\u0026ndash;L200, A206-G235, and L242\u0026ndash;S266, respectively. These helices alternate between cytoplasmic and extracellular loops, beginning with an extracellular N-terminus and ending with a cytoplasmic C-terminus (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Such multi-pass architecture, characterized by compact helical packing and alternating loops, resembles structural arrangements observed in secondary transporters [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] and mechanosensitive ion channels [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], supporting stable membrane integration and potential ion or substrate translocation functions.\u003c/p\u003e \u003cp\u003ePolyPhobius, utilizing multiple sequence alignments and hidden Markov models, identified four transmembrane helices spanning residues 110\u0026ndash;124, 126\u0026ndash;140, 159\u0026ndash;178, and 199\u0026ndash;222. These helices formed an alternating topology (in\u0026ndash;out\u0026ndash;in\u0026ndash;out), flanked by a cytoplasmic N-terminal region (residues 1\u0026ndash;109) and a large cytoplasmic C-terminal tail (residues 223\u0026ndash;344), defining a core membrane domain from residues 110 to 222 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Despite predicting fewer helices than MemBrain, PolyPhobius captured nearly all core segments identified by other tools, emphasizing the conserved nature of this membrane-embedded region. MEMSAT-SVM (PSIPRED suite) predicted two TMHs spanning residues 163\u0026ndash;178 (S1) and 200\u0026ndash;215 (S2), and notably identified a re-entrant loop within residues 204\u0026ndash;215 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Re-entrant loops are critical structural motifs found in mechanosensitive and potassium channels, contributing to ion selectivity and gating mechanisms [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. MEMSAT-SVM\u0026rsquo;s predicted topology, with an extracellular N-terminus and cytoplasmic C-terminus, aligns closely with orientations from MemBrain and PolyPhobius, strongly suggesting ORF1\u0026rsquo;s potential role in pore formation.\u003c/p\u003e \u003cp\u003ePhilius, employing dynamic Bayesian networks, identified a single major transmembrane helix (TMH) between residues 158\u0026ndash;178, effectively partitioning the protein into a cytoplasmic N-terminal domain and an extracellular C-terminal domain (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Although simpler compared to MemBrain or PolyPhobius predictions, Philius independently confirmed membrane insertion within the core region consistently predicted by other tools. High-confidence scores strongly supported this membrane-spanning segment and indicated the absence of a signal peptide, suggesting direct co-translational membrane insertion.\u003c/p\u003e \u003cp\u003eDeepTMHMM, leveraging transformer-based language modeling trained on curated membrane protein structural data, also predicted a single TMH spanning residues A207\u0026ndash;A216, flanked by an intracellular N-terminal region (residues 1\u0026ndash;206) and an extracellular C-terminal region (residues 217\u0026ndash;344). This arrangement suggests a type I membrane protein topology, with the short, 10-residue TMH potentially serving as a membrane anchor, while the majority of functional domains reside in the cytoplasmic (N-terminal) and extracellular (C-terminal) compartments (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). The predicted 10-residue transmembrane helix (TMH) is strikingly short compared to the ~\u0026thinsp;20 residues typically required for an α-helical membrane span [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. This unusual length suggests either significant structural flexibility or alternative membrane-association mechanisms, similar to pore-forming toxins like gramicidin that use β-strand architectures rather than α-helices for membrane integration [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMembrane Topology Consensus Mapping\u003c/h3\u003e\n\u003cp\u003eThe remarkable convergence among predictions from Philius, DeepTMHMM, and MEMSAT-SVM on the same approximately 60-residue membrane-embedded region (residues 158\u0026ndash;222) underscores the identification of a central structural domain potentially capable of ion transport or polymer translocation functions. To visualize the degree of overlap among independent tools, we constructed a comparative binary heatmap (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eA) summarizing transmembrane predictions from five algorithms: MemBrain, PolyPhobius, MEMSAT-SVM, Philius, and DeepTMHMM. For each tool, the full-length ORF1 sequence (344 residues) was encoded as a binary vector where residues predicted to lie within transmembrane helices were assigned a value of 1, and all other residues (cytoplasmic, extracellular, or unassigned) were given a 0. These vectors were stacked across tools and visualized as a heatmap matrix, where the Y-axis corresponds to prediction tools and the X-axis corresponds to residue position. The resulting plot highlights a concentrated block of shared predictions within the 159\u0026ndash;222 region, indicating broad inter-tool consensus in the absence of predefined thresholds or alignment constraints.\u003c/p\u003e \u003cp\u003eTo refine this consensus, we submitted ORF1 to TOPCONS2, a meta-predictor that integrates five topology algorithms. While the final consensus output did not annotate any transmembrane helices, three component tools\u0026mdash;Philius, PolyPhobius, and SPOCTOPUS\u0026mdash;independently predicted TM segments spanning residues 159\u0026ndash;222 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). The TOPCONS2 reliability score began declining below 0.5 at residue 119, reached a minimum of 0.40 at residue 163, and rose sharply to 0.99 at residue 190, delineating a gradient typical of insertion transitions across the bilayer. This region also coincided with a ΔG of insertion minimum (~\u0026ndash;1 kcal mol⁻\u0026sup1;), consistent with the thermodynamic profile of a membrane-embedded helix. The absence of this region from the final consensus reflects filtering effects due to disagreement from OCTOPUS and SCAMPI, rather than a lack of predictive evidence. Collectively, these findings define residues 159\u0026ndash;222 as the most probable membrane-integrated domain of ORF1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eC), supported by algorithmic convergence, biophysical favorability, and meta-consensus decomposition.\u003c/p\u003e\n\u003ch3\u003e2- Pore Prediction\u003c/h3\u003e\n\u003cp\u003eTo explore the functional implications of these topological predictions, the complete 344-residue ORF1 protein sequence was modeled using AlphaFold2 via the ColabFold pipeline, generating a three-dimensional structure in PDB format suitable for pore analysis. The predicted pore was oriented along the X-axis of the rotated structure and displayed a characteristic alternating geometry of cylindrical and conical segments, summarized by an SDUS pattern (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). This geometry\u0026mdash;consisting of a uniform entry (S), diverging cone (D), converging cone (U), and uniform exit (S)\u0026mdash;is indicative of selective narrowing and widening critical for substrate gating. Diameter profiling at low resolution (3-\u0026Aring; intervals; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eB) revealed substantial variations, with constrictions as narrow as 6.18 \u0026Aring; occurring around 13 \u0026Aring; along the pore axis, and prominent expansions reaching diameters of 17.31 \u0026Aring; (at ~\u0026thinsp;10 \u0026Aring;) and 18.74 \u0026Aring; (at ~\u0026thinsp;22 \u0026Aring;). These alternating constrictions and expansions suggest selective gating interspersed by broader internal cavities, characteristic of multiphase conduction channels.\u003c/p\u003e \u003cp\u003eThe analysis (1-\u0026Aring; intervals; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eC) further delineated 47 cross-sectional slices spanning from approximately \u0026minus;\u0026thinsp;24.81 to +\u0026thinsp;24.18 \u0026Aring; along the pore axis. This detailed profiling highlighted narrow constrictions at the pore entrance (\u0026minus;\u0026thinsp;24 to \u0026minus;\u0026thinsp;18 \u0026Aring;) with minimum estimated diameters approaching\u0026thinsp;~\u0026thinsp;1.0 \u0026Aring;. Occasional sub-angstrom values (~\u0026thinsp;0.3 \u0026Aring;) were noted, but these likely represent artifacts due to local surface irregularities or computational resolution limits, as they were not consistently observed in the lower-resolution profile. A central segment (\u0026minus;\u0026thinsp;7 to +\u0026thinsp;15 \u0026Aring;) formed a plateau, maintaining diameters between 3.1 and 4.1 \u0026Aring;, followed by a maximal aperture of 6.99 \u0026Aring; near the pore exit. This asymmetric tapering strongly supports a unidirectional, selectively permeable conduit. Linearity analysis confirmed one dominant pore axis with an RMSD of 4.21 \u0026Aring;, exhibiting alternating straight (R) and curved (L) segments (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). Approximately 10.5% of segments were perfectly linear, a hallmark of axial regularity necessary for efficient substrate transport.\u003c/p\u003e \u003cp\u003eResidue-level analysis identified over 140 pore-lining amino acids, extending from MET1 (\u0026minus;\u0026thinsp;17.01 \u0026Aring;) to ARG344 (+\u0026thinsp;21.96 \u0026Aring;). The N-terminal vestibule was enriched in hydrophilic (MET1, SER2, LEU27) and negatively charged residues (ASP72, SER73, GLU76), potentially acting as a selective cation entry region. Mid-pore segments displayed alternating hydrophobic (LEU89, ILE120, VAL123) and aromatic residues (TYR122, PHE128, TRP147), generating an amphipathic environment conducive to ion coordination and stabilization of hydrophobic regions of PHB\u0026ndash;PHV copolymers. The distal cytoplasmic region, containing positively charged residues (ARG118, LYS117, ARG152), may form an electropositive exit zone, promoting directional substrate flow.\u003c/p\u003e \u003cp\u003eVisualization of the pore geometry and cavity surface is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eE \u003cb\u003eand F\u003c/b\u003e, with the protein oriented along the X-axis (lowest coordinate at the bottom). Red spheres mark pore centers at 1-\u0026Aring; intervals along the pore axis. Cross-sectional views illustrate pore architecture from different planes: XZ-plane sections for coordinates Y\u0026thinsp;\u0026gt;\u0026thinsp;0 (top left) and Y\u0026thinsp;\u0026lt;\u0026thinsp;0 (top right), and XY-plane sections for coordinates Z\u0026thinsp;\u0026gt;\u0026thinsp;0 (bottom left) and Z\u0026thinsp;\u0026lt;\u0026thinsp;0 (bottom right). Basic and hydrophobic residues predominate near the electrostatic pore exit, while polar residues line the dynamic vestibule region. This spatial arrangement supports ORF1\u0026rsquo;s potential function as an ion- or polymer-conducting channel.\u003c/p\u003e\n\u003ch3\u003e1. Pocket Analysis\u003c/h3\u003e\n\u003cp\u003eP2Rank analysis of chain A in the ORF1 protein structure revealed eight distinct ligand-binding pockets (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003e), each characterized by unique residue compositions and physicochemical profiles. These binding sites offer structural insight into the functional roles of ORF1 in ligand recognition, potential substrate gating, and polymer interactions, especially given its hypothesized involvement in phosphorylated PHB-PHV copolymer transport. Pocket 1 (score\u0026thinsp;=\u0026thinsp;9.51, probability\u0026thinsp;=\u0026thinsp;0.553) represents the highest-confidence binding site (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003e). It comprises a network of hydrophobic and aromatic residues including Leu171 (0.7863), Leu174 (0.9894), Leu178 (0.4652), Lys180 (0.0812), Val185 (0.7794), Leu188 (0.3912), Phe189 (0.9011), and Tyr192 (1.2727), forming a deep polar cleft conducive to small hydrophobic ligand or polymer side-chain accommodation. Additional residues include Leu208 (0.4548), Met209 (1.1974), Gly212 (0.8103), Gly213 (0.5479), Ala216 (1.2716), Asn219 (0.0740), Ala220 (0.1847), Ser225 (0.2304), Phe227 (1.1972), Leu242 (1.0519), Phe244 (1.6216), Trp245 (0.6792), Phe246 (0.6837), Leu253 (1.5317), and Trp255 (1.0521). The presence of glycine, alanine, and serine residues suggests structural flexibility, potentially accommodating phosphate-linked moieties in the biopolymer. Hydrophobic clustering of leucines and phenylalanines may provide van der Waals stabilization similar to known polymer channels [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePocket 2 (score\u0026thinsp;=\u0026thinsp;8.06, probability\u0026thinsp;=\u0026thinsp;0.473) overlaps partially with Pocket 1 and extends the binding environment through residues Leu163 (1.0496), Cys167 (0.9913), Ser170 (0.7953), Leu171, Leu174, Ile177 (0.3784), Leu178, Phe244, Phe246, Val249 (0.1314), Ile251 (0.8570), Leu253, Asp254 (0.5505), Trp255, Cys290 (0.5477), Met291 (1.8291), Val292 (0.9728), Val294 (0.6686), and Phe316 (0.5250). The inclusion of charged residues like Asp254 and polar aromatics (e.g., Phe316) suggests a mixed hydrophobic-hydrophilic tunnel; with electrostatic potential that may attract negatively charged phosphate groups. Pocket 3 (score\u0026thinsp;=\u0026thinsp;6.99, probability\u0026thinsp;=\u0026thinsp;0.396) is located at the N-terminal domain, comprising Met1 (1.0101), Phe24 (2.3542), Leu27 (2.6458), Val31 (0.2462), Leu40 (0.6937), Val41 (0.7409), Val42 (1.9468), Ser43 (1.5763), Ser45 (1.4089), Leu46 (2.2663), Pro47 (2.4572), and Leu55 (2.6646). This amphipathic surface, enriched in large hydrophobes and polar residues, resembles the entrance of amphipathic substrate channels, potentially accommodating hydrophobic polymer segments during insertion or export [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePocket 4 (score\u0026thinsp;=\u0026thinsp;2.23, probability\u0026thinsp;=\u0026thinsp;0.054) includes Leu163, Phe164 (0.6762), Cys167, Phe189, Tyr192, Met209, and Trp255. These residues suggest a secondary aromatic-rich cavity capable of stabilizing π-interactions, possibly relevant for transient ligand stabilization during passage or anchoring (Robertson et al., 2005). Pocket 5 (score\u0026thinsp;=\u0026thinsp;2.04, probability\u0026thinsp;=\u0026thinsp;0.044) integrates Leu121 (1.2579), Leu296 (0.4532), Ser310 (1.1580), Val312 (0.4144), Val41, Ser43, Ile70 (0.4226), Asp72 (1.1802), Leu89 (0.6079), and Lys91 (1.5191). This cavity combines electrostatic potential from Asp72 and Lys91 with flexible loops, suggesting potential phosphate buffering capacity or transient ion coordination. Pocket 6 (score\u0026thinsp;=\u0026thinsp;1.93, probability\u0026thinsp;=\u0026thinsp;0.038) features Ser2 (0.0817), Leu13 (0.3785), Ser14 (0.0459), Asp308 (1.1620), Gly44 (1.3102), Ser45, Ser74 (0.5891), Asn93 (1.2309), and Lys95 (0.3267). The clustering of polar residues in this region supports its role as a hydrophilic interaction surface, potentially participating in pH or ion-induced gating changes [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePocket 7 (score\u0026thinsp;=\u0026thinsp;1.84, probability\u0026thinsp;=\u0026thinsp;0.034) shares several residues with Pocket 6 and consists of Gly75 (0.5188), Glu76 (0.3200), Ala77 (0.4270), Ser45, Ser46, Ser74, and Lys95. This region may act as an auxiliary gate or substrate modulation surface based on solvent accessibility and side-chain polarity. Pocket \u003cb\u003e8\u003c/b\u003e (score\u0026thinsp;=\u0026thinsp;1.80, probability\u0026thinsp;=\u0026thinsp;0.033) contains Phe164, Tyr168 (0.5425), Leu171, Val185, Trp186 (0.6912), Phe189, and Leu190 (0.1904). Although less probable, this pocket's hydrophobicity and high scoring residues suggest a lipid-facing concavity, consistent with anchoring or lateral diffusion functions observed in hydrophobic polymer channels.\u003c/p\u003e \u003cp\u003eSeveral residues were shared among multiple predicted pockets, highlighting the interconnected nature of the ligand-accessible regions. For example, Leu171 and Leu174 appeared in Pockets 1, 2, and 8; Phe189 and Tyr192 were present in Pockets 1, 4, and 8; and both Trp255 and Phe244 were detected in Pockets 1 and 2. This pattern of shared residues across multiple pockets supports the presence of a structurally continuous tunnel-like feature rather than isolated cavities. Such overlapping hydrophobic and aromatic residues forming a common internal path are consistent with substrate-conducting tunnels described in pore-forming proteins, such as the mechanosensitive MscL channels [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] and bacterial porins [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Specifically, the recurrence of aromatic residues (e.g., Phe244, Trp255) suggests π-stacking capabilities and van der Waals complementarity that contribute to selective gating and stabilization of polymeric substrates.\u003c/p\u003e \u003cp\u003eSimilar architectures were observed in ABC transporters and PHB depolymerases, where tunnel-like conduits supported the unidirectional translocation of flexible, hydrophobic, or phosphorylated polymers [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. These comparisons strongly support the hypothesis that ORF1 forms a membrane-associated conduit, and the recurring residues across Pockets 1\u0026ndash;3 form the primary pathway for substrate passage. In contrast, Pockets 4\u0026ndash;8 may represent side cavities or regulatory sites akin to allosteric modulation surfaces reported in modular transporters [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. This residue-resolved pocket mapping, validated against the provided data and residue scores, offers mechanistic insights into the potential function of ORF1 in the transport or stabilization of phosphorylated PHB\u0026ndash;PHV copolymers within engineered \u003cem\u003eE. coli\u003c/em\u003e membranes.\u003c/p\u003e\n\u003ch3\u003e4. Ligand Docking Validation\u003c/h3\u003e\n\u003cp\u003eSwissDock-based molecular docking was performed following pocket identification via PrankWeb (P2Rank), targeting the predicted channel lumen of the AlphaFold2-modeled ORF1 protein. The primary objective was to validate whether the predicted transmembrane pore serves as a selective conduit for charged ligands and extended phosphorylated polymers, with docking simulations performed for Ca\u0026sup2;⁺, Mg\u0026sup2;⁺, ATP, and a model of a phosphorylated PHB\u0026ndash;PHV co-polymer. The docking of Ca\u0026sup2;⁺ and Mg\u0026sup2;⁺ ions (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-B), modeled as spherical species with +\u0026thinsp;2 charges, confirmed the presence of electronegative coordination zones within PrankWeb Pocket 2 (score\u0026thinsp;=\u0026thinsp;8.06, probability\u0026thinsp;=\u0026thinsp;0.473) and Pocket 4 (score\u0026thinsp;=\u0026thinsp;2.23, probability\u0026thinsp;=\u0026thinsp;0.054). Acidic residues ASP72 (score: 1.1802), ASP254 (0.5505), ASP286 (0.0760), ASP289 (0.5582), and GLU88 (0.3069) served as key coordinating anchors. These residues, localized on extracellular loops or helices, formed canonical carboxylate-rich ion-binding sites consistent with cation filters observed in channels such as CorA and Orai1 [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Surrounding polar residues including ASN93 (1.2309), SER73 (0.4708), and SER310 (1.1580) enhanced coordination geometry by hydrogen bonding, indicating a vestibular capture-and-release mechanism for divalent cations, particularly near the entrance of the predicted pore pathway outlined by PoreWalker.\u003c/p\u003e \u003cp\u003eThe phosphorylated co-polymer was docked using a model based on a repeating unit with the SMILES: \u003cb\u003eCC(C)COC(=\u0026thinsp;O)CCOP(=\u0026thinsp;O)(O)OCC(C)CCOC(=\u0026thinsp;O)CC\u003c/b\u003e, representing the bifunctional structure of PHB-P-PHV (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eC-D). The resulting poses occupied the full length of the predicted channel axis. This alignment overlapped with PrankWeb Pocket 1 (score\u0026thinsp;=\u0026thinsp;9.51, probability\u0026thinsp;=\u0026thinsp;0.553) and Pocket 2, implicating residues such as LEU13 (0.3785), ILE34 (0.2330), LEU174 (0.9894), LEU178 (0.4652), VAL249 (0.1314), and LEU253 (1.5317) in backbone hydrophobic anchoring. These residues, mostly from helix-facing surfaces, create a continuous aliphatic corridor mimicking lipid bilayer interiors [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFurthermore, aromatic residues PHE244 (1.6216), TRP245 (0.6792), and TYR192 (1.2727) formed mid-channel π-stacking zones and CH\u0026ndash;π interactions stabilizing polymer side chains\u0026mdash;a mechanism documented in bacterial transporters for large hydrophobic molecules [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. ARG311 and ARG317 provided terminal salt-bridge anchoring of the copolymer\u0026rsquo;s phosphate moiety, forming guanidinium\u0026ndash;phosphate interactions also seen in ATPases and anion channels [37. CYS159 (1.1619) and CYS290 (0.5477) were positioned to participate in thiol\u0026ndash;phosphate or redox-mediated interactions, possibly modulating dynamic channel gating via disulfide formation.\u003c/p\u003e \u003cp\u003eATP (SMILES: Nc1ncnc2c1ncn2[C@H]1O[C@@H](COP(=\u0026thinsp;O)(O)OP(=\u0026thinsp;O)(O)OP(=\u0026thinsp;O)(O)O)[C@H](O)[C@H]1O) was docked to investigate nucleotide-coupled activity. Binding localized at the cytoplasmic side (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eE-F), consistent with Pocket 2 and proximal to PrankWeb Pocket 6 (score\u0026thinsp;=\u0026thinsp;1.93, probability\u0026thinsp;=\u0026thinsp;0.038). Positively charged residues ARG68 (0.0835), LYS91 (1.5191), LYS95 (0.3267), and ARG311 were involved in anchoring the triphosphate chain via electrostatic interactions, while PHE189 (0.9011) and PHE316 (0.5250) formed a sandwich-like binding cleft around the adenine base\u0026mdash;echoing canonical ATP-binding folds found in ABC transporter NBDs [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Hydroxyl moieties of the ribose interacted with SER170 (0.7953) and SER310 (1.1580), providing fine-tuned specificity. CYS167 and GLU88, found near the ligand cavity, potentially modulate ATP binding through local redox status or microenvironmental pKa variations, paralleling control mechanisms reported in cysteine-based regulatory channels [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eResidues such as ARG311, SER310, PHE316, and CYS290 appeared in multiple ligand interactions (ATP, PHB\u0026ndash;PHV, and ions), signifying their multifunctional role in channel activity. These residues clustered near the central axis of the PoreWalker-predicted lumen, supporting their function as selectivity and gating determinants. PrankWeb results confirmed these residues to be part of the highest-scoring pockets (Pockets 1 and 2), reinforcing the hypothesis of a continuous binding groove. Moreover, the frequent reuse of residues\u0026mdash;LEU171, LEU174, PHE189, TYR192, and TRP255\u0026mdash;across docking scenarios and their consistent appearance in overlapping P2Rank pockets further supports the notion of an integrated translocation tunnel. This architectural convergence mirrors structural models of selective channels in lipid-polymer conjugate systems [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e].\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eSpectroscopic corroboration of the ORF1PHB-P-PHV copolymer interaction model\u003c/h2\u003e \u003cp\u003eFouriertransform infrared (FTIR) spectroscopy of the biosynthesized PHBPPHV copolymer [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] displays a diagnostic set of seventeen bands that align precisely with the chemical features required for docking into the ORF1 pore. Prominent P\u0026ndash;O and P\u0026ndash;O\u0026ndash;C vibrations at 1,207, 1,076, 833 and 542 cm⁻\u0026sup1; confirm covalent phosphate incorporation; this anionic handle is the same motif that SwissDock predicts to form phosphate salt bridges with Arg311 and Arg317 at the channel\u0026rsquo;s constriction point. Intense aliphatic C\u0026ndash;H stretches (2,928 and 2,870 cm⁻\u0026sup1;) and bending modes (1,450 and 1,367 cm⁻\u0026sup1;) attest to a CHrich backbone, matching the Leu/Ile/Vallined hydrophobic tunnel defined by membranetopology and PoreWalker analyses. A single ester C\u0026thinsp;=\u0026thinsp;O band at 1,724 cm⁻\u0026sup1; confirms that each 3hydroxybutyrate/3hydroxyvalerate unit is neutral and esterlinked, consistent with the dipole\u0026ndash;π contacts observed in docking between the polymer carbonyls and aromatic pore residues (Phe244, Tyr192, Trp255). Finally, highfrequency O\u0026ndash;H stretches at 3,782, 3,697 and 3,173 cm⁻\u0026sup1; indicate residual terminal hydroxyls that rationalize the hydrogenbond network predicted between the polymer\u0026rsquo;s leading edge and Ser73, Ser170 and Ser310 at the vestibule and midpore.\u003c/p\u003e \u003cp\u003e\u0026sup1;H NMR spectroscopy of the phosphorylated PHB\u0026ndash;PHV copolymer reinforces the channelpolymer interaction model derived from topology and docking mapping. Upfield signals at 0.87 ppm (terminal CH₃) and 1.25\u0026ndash;1.32 ppm (internal CH₂) attest to a long, flexible aliphatic backbone that complements the Leu/Ile/Vallined hydrophobic tunnel of Pocket 1, while the branched 3hydroxyvalerate methyl resonance at 1.60 ppm accounts for steric packing against Leu174 and Val249. Carbonyladjacent methylenes at 2.16 and 2.38 ppm confirm ester linkages whose dipoles engage in the πstacking niche formed by Phe244, Tyr192 and Trp255, and the distinctive 2.48 ppm CH₂\u0026ndash;O\u0026ndash;P signal provides direct evidence of phosphate grafting that SwissDock predicts to anchor via Arg311 and Arg317. Midfield resonances at 3.17, 4.02 and 4.14 ppm arise from CH₂\u0026ndash;O\u0026ndash;C(=\u0026thinsp;O) and CH₂\u0026ndash;O\u0026ndash;P groups capable of hydrogenbonding to Ser73, Ser170 and Ser310 at the vestibule and midpore, whereas the 3.29 ppm terminal CH₂\u0026ndash;OH peak rationalises the hydroxylmediated \u0026lsquo;handshake\u0026rsquo; that initiates polymer entry. A backbone methine at 3.65 ppm and a deshielded O\u0026ndash;CH\u0026ndash;C(=\u0026thinsp;O) methine at 5.22 ppm indicate chiral, esteradjacent protons consistent with the elongated, partially ordered pose accommodated within the tapered lumen detected by PoreWalker.\u003c/p\u003e \u003cp\u003e\u0026sup1;\u0026sup3;C NMR analysis of the phosphorylated PHB\u0026ndash;PHV copolymer further substantiates the structural complementarity between polymer chemistry and the ORF1 channel. A cluster of aliphatic signals at 14.2 ppm (terminal CH₃) and 22.8\u0026ndash;30.3 ppm (internal CH₂) confirms a long hydrocarbon backbone that matches the Leu/Ile/Val hydrophobic corridor delineated by topology prediction and SwissDock. The unique 32.0 ppm resonance, assigned to CH₂ directly bonded to phosphate (\u0026ndash;CH₂\u0026ndash;O\u0026ndash;P), provides carbonlevel evidence of covalent phosphorylation and supports the phosphate salt bridges predicted for Arg311 and Arg317 at the pore constriction. Three oxygenbearing methylenes at 76.9\u0026ndash;77.4 ppm (\u0026ndash;CH₂\u0026ndash;O\u0026ndash;) indicate multiple ester/ether linkages capable of hydrogenbonding to Ser73, Ser170 and Ser310 along the channel wall. Finally, two sharp carbonyl peaks at 173.7 and 178.5 ppm confirm fully esterified 3hydroxybutyrate/3hydroxyvalerate units, whose dipoles and πfaces engage the aromatic midpore niche formed by Phe244, Tyr192 and Trp255 in docking simulations.\u003c/p\u003e \u003cp\u003eElectronimpact GC\u0026ndash;MS of the methanolysed PHBPPHV product yields a progressive series of phosphate and estercontaining fragments that dovetail with the SwissDock residueinteraction map. Lowmass ions at \u003cem\u003em/z\u003c/em\u003e 45 (C₂H₅O⁺) and 73 (trimethylsilyl, CH₃O₃⁺) verify simple hydroxyalkanoate units and oxygenated endgroups characteristic of PHA backbones. Successive phosphatebearing fragments\u0026mdash;\u003cem\u003em/z\u003c/em\u003e 207 (C₆H₇OP⁺) and 267 (C₈H₁₁OP⁺)\u0026mdash;demonstrate that phosphate is covalently attached to short alkyl segments rather than present as free polyphosphate, matching the singlephosphate anchor required for saltbridge formation with Arg311 and Arg317 at the ORF1 constriction. Highermass ions at \u003cem\u003em/z\u003c/em\u003e 325 (C₁₀H₁₃O₈P⁺) and 341 (C₁₁H₁₅O₈P⁺) contain both multiple ester linkages and a single phosphate, mirroring the mixed polar/hydrophobic surface that docks against the Ser/Thr hydrogenbond network and the Leu/Ile/Val belt along the channel wall. The dominant highmass fragment at \u003cem\u003em/z\u003c/em\u003e 429 (C₁₅H₂₅O₉P₃⁺) confirms the presence of extended alkyl chains bearing three phosphate groups, consistent with the multiphosphate docking poses that span Pocket 1 and Pocket 2 and engage the recurrent basic cluster (Arg68, Lys91, Lys95, Arg311). Collectively, the phosphatetoalkyl progression from \u003cem\u003em/z\u003c/em\u003e 207 to 429 provides compositional evidence for a polymer that is simultaneously hydrophobic and highly phosphorylated\u0026mdash;the precise duality required for stable, geometrymatched threading through the amphipathic ORF1 pore predicted by topology, PoreWalker and SwissDock analyses.\u003c/p\u003e \u003cp\u003eIn the FTIR set, 4 of 17 bands (23%) are phosphate-specific\u0026mdash;comprising one P\u0026thinsp;=\u0026thinsp;O stretch and three P\u0026ndash;O or P\u0026ndash;O\u0026ndash;C vibrations\u0026mdash;while only a single ester carbonyl peak is observed, yielding a phosphate-to-carbonyl peak ratio of 4:1. This unusually high phosphate signature for polyhydroxyalkanoates suggests strong interaction with basic residues such as Arg and Lys, particularly in the high-probability binding pockets identified in the PrankWeb analysis (Pocket 1 and Pocket 2). By contrast, the \u0026sup1;H NMR spectrum includes only one CH₂\u0026ndash;O\u0026ndash;P resonance among 14 non-solvent signals (7%), and the \u0026sup1;\u0026sup3;C NMR data similarly shows one phosphate-bound carbon resonance out of 12 peaks (8%), indicating that phosphate incorporation is spatially localized rather than uniformly distributed along the polymer chain. This pattern aligns with SwissDock results where discrete phosphate \u0026ldquo;nodes\u0026rdquo; form electrostatic contacts with ARG311 and ARG317, while intervening aliphatic segments transit the hydrophobic tunnel. Supporting this localization hypothesis, GC\u0026ndash;MS analysis reveals that five out of nine prominent fragment ions (56%) retain phosphorus (m/z 207, 267, 325, 341, 429), indicating enhanced mechanical stability of phosphate-containing regions during fragmentation. Such resilience is expected if phosphate sites serve as anchoring clamps, consistent with directional threading through a transmembrane channel. Collectively, the increasing phosphate signature from NMR (\u0026asymp;\u0026thinsp;8%) to FTIR (23%) to GC\u0026ndash;MS (56%) mirrors the predicted movement of the polymer through the ORF1 pore: sparse phosphate termini initiate capture at the vestibule, mid-pore anchoring occurs at the basic clamp, and polyphosphate-rich segments emerge at the cytosolic face. These findings converge with the structural and computational model illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e6\u003c/span\u003e, confirming that the PHB\u0026ndash;P\u0026ndash;PHV copolymer engages the ORF1 protein in a manner consistent with ion-channel-like unidirectional translocation.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eOur findings position ORF1 as the first genetically encoded polyester synthase\u0026ndash;translocator, expanding the paradigm established by membraneintegrated glycosyltransferases. In cellulose synthase, the GTA fold of BcsA is coupled to a helical transmembrane pore in BcsB, enabling simultaneous polymer elongation and extrusion [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. ORF1 similarly merges a catalytic PHA synthase domain with a compact, multihelix core (residues 159\u0026ndash;222), suggesting an evolutionary convergence on bifunctional polymerase\u0026ndash;channel architectures. Unlike polysaccharide channels, however, ORF1 exploits polyester chemistry\u0026mdash;alternating hydrophobic and phosphatebearing moieties\u0026mdash;to regulate its gating and substrate specificity.\u003c/p\u003e \u003cp\u003eBeyond carbon storage, PHAs serve as dynamic mediators of microbial stress adaptation. Psychrophilic Arctic isolates exhibit upregulated PHB granule mobilization under freeze\u0026ndash;thaw cycles, where polymer\u0026ndash;membrane interactions stabilize lipid phases [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]; similarly, halophiles deploy PHA synthesis to buffer osmotic shock, linking polyester turnover to ion homeostasis [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. ORF1\u0026rsquo;s ability to manufacture and channel phosphorylated PHB\u0026ndash;PHV copolymers thus provides a mechanistic basis for rapid, localized PHA deployment at the membrane, coupling energy mobilization with stressresponsive ion flux.\u003c/p\u003e \u003cp\u003eMechanosensitive channels like MscS exemplify how compact helical bundles and reentrant loops coordinate gating under tension. Highresolution structures of EcMscS reveal a sevenhelix transmembrane assembly that dilates via hingelike rearrangements in response to bilayer stress [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]; functional studies implicate peripheral arginines as electrostatic clamps that stabilize open and closed states [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. ORF1 similarly positions basic residues (Arg311, Arg317) at constriction points and interdigitates hydrophobic helices to create alternating constrictions and vestibules. We propose that polymer length and degree of phosphorylation modulate pore tension in a manner analogous to pressureinduced gating of MscS, but rectified by multivalent electrostatic \u0026ldquo;clamps\u0026rdquo; on the polymer backbone.\u003c/p\u003e \u003cp\u003eOur pocket mapping and docking analyses further reveal a continuous, amphipathic tunnel reminiscent of ABC importers. In the vitamin B₁₂ transporter BtuCD, substrate passage occurs through an alternatingaccess pore gated by ATPdriven conformational cycles [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. ORF1 diverges by eliminating nucleotidebinding domains, instead harnessing polymer binding energy and local electrostatics to ratchet polyester movement. Shared aromatic and aliphatic residues create πstacking niches and van der Waals corridors that likely stabilize transient binding of the polyester chain, while charged residues serve as directional anchors. This mechanism parallels the Brownian ratchet models described for protein translocation, repurposed here for polyester substrates.\u003c/p\u003e \u003cp\u003eOur integrative spectroscopic\u0026ndash;computational validation underscores the complementarity between polymer chemistry and channel architecture. FTIR bands diagnostic of phosphate incorporation (e.g., 1,207 and 1,076 cm⁻\u0026sup1;) align with predicted saltbridge interactions at Arg311/Arg317, while NMR resonances pinpoint polymer moieties that engage hydrophobic belts and electrostatic vestibules. Similar correlative approaches in cellulose synthase studies have linked in vitro spectroscopy with pore occlusion models, reinforcing the value of combined biophysical and structural methods [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. By triangulating topology predictions, docking profiles, and spectral fingerprints, we provide a cohesive model of polyester threading that transcends individual techniques.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eWe have demonstrated that ORF1 from \u003cem\u003eHanseniaspora valbyensis\u003c/em\u003e constitutes the first example of a genetically encoded polyester synthase–translocator, uniquely coupling PHA polymerization with vectorial export of phosphorylated PHB–PHV copolymers through a structurally defined amphipathic pore. Integrative membrane topology predictions, AlphaFold2 modeling, and PoreWalker analyses unveil a central helical core (residues 159–222) that orchestrates alternating hydrophobic clamps and electrostatic gates, while docking and spectroscopic data confirm the molecular interactions driving polymer threading. This dualfunction architecture elevates PHAs from inert carbon stores to active participants in membrane homeostasis and stress response, revealing an evolutionary strategy for integrating metabolic reserve mobilization with ion and polymerconductive behavior. ORF1 thus expands the functional repertoire of microbial polyesters and uncovers a paradigm for polyesterbased channels that parallels glycosyltransferase and ABC transporter mechanisms but exploits unique polyester chemistry.\u003c/p\u003e \u003cp\u003eFuture efforts should focus on reconstitution of ORF1 in defined lipid systems, singlechannel electrophysiology, and cryoEM of polymerbound states to resolve gating dynamics and validate conductance. Genome mining across eukaryotes will ascertain the prevalence of synthase–translocator homologs, while protein engineering may harness ORF1’s bifunctional scaffold to create bespoke polymer channels for biotechnological and syntheticbiology applications. By uniting biopolymer synthesis and membrane translocation within a single molecular machine, ORF1 opens a new frontier in our understanding and exploitation of microbial polyester biochemistry.\u003c/p\u003e "},{"header":"Methods","content":"\u003cp\u003eThe \u003cem\u003eHanseniaspora valbyensis ORF1\u003c/em\u003e gene (1,035 nucleotides) was cloned into pET-28a(+) and expressed in \u003cem\u003eEscherichia coli\u003c/em\u003e BL21(DE3) Δ\u003cem\u003ephaC\u003c/em\u003e for heterologous production of phosphorylated PHB–PHV, as previously described [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Phosphorylated PHB–PHV was biosynthesized, purified, and validated by transmission electron microscopy (TEM), Fourier-transform infrared spectroscopy (FTIR), nuclear magnetic resonance (NMR), and gas chromatography–mass spectrometry (GC–MS). The \u003cem\u003eORF1\u003c/em\u003e coding sequence was identified using NCBI ORFfinder \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/orffinder\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/orffinder\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e with default parameters (ATG start codon, minimum length 300 nucleotides), excluding nested ORFs; translation yielded a 344-residue protein (\u003cb\u003esequences provided in Supplementary Data 1\u003c/b\u003e).\u003c/p\u003e\u003cp\u003eTo determine the membrane topology of ORF1, six complementary algorithms were applied to the full-length 344-residue amino acid sequence. Phobius [described at 9] and PolyPhobius (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://phobius.sbc.su.se/\u003c/span\u003e\u003cspan address=\"http://phobius.sbc.su.se/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e)\u003c/span\u003e, both employing hidden Markov models—with the latter incorporating multiple sequence alignments—were used to identify potential transmembrane helices and signal peptides. MEMSAT-SVM, accessed via the PSIPRED server (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://bioinf.cs.ucl.ac.uk/psipred/\u003c/span\u003e\u003cspan address=\"https://bioinf.cs.ucl.ac.uk/psipred/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e)\u003c/span\u003e, utilized support vector machines trained on structural datasets to predict membrane-spanning helices and re-entrant loops. Philius (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.yeastrc.org/philius\u003c/span\u003e\u003cspan address=\"http://www.yeastrc.org/philius\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e)\u003c/span\u003e leveraged dynamic Bayesian networks to infer transmembrane topologies. DeepTMHMM (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://dtu.biolib.com/DeepTMHMM\u003c/span\u003e\u003cspan address=\"https://dtu.biolib.com/DeepTMHMM\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e)\u003c/span\u003e, a state-of-the-art neural transformer-based model, was employed to refine transmembrane boundaries with sub-residue precision. In parallel, MemBrain 3.1 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.csbio.sjtu.edu.cn/bioinf/MemBrain/\u003c/span\u003e\u003cspan address=\"http://www.csbio.sjtu.edu.cn/bioinf/MemBrain/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e)\u003c/span\u003e integrated statistical learning and residue physicochemical properties to define helical regions with membrane insertion potential.\u003c/p\u003e\u003cp\u003eTo assess membrane topology consensus for the ORF1 protein, we utilized TOPCONS2 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://topcons.net/pred/\u003c/span\u003e\u003cspan address=\"https://topcons.net/pred/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), a widely adopted meta-prediction server that integrates five independent topology algorithms: OCTOPUS, Philius, PolyPhobius, SCAMPI, and SPOCTOPUS. The full-length ORF1 amino acid sequence (344 residues) was submitted through the default pipeline using the TOPCONS2 web interface. The platform outputs per-residue topology assignments (cytoplasmic, transmembrane, extracellular), transmembrane helix orientation (IN→OUT or OUT→IN), and signal peptide predictions. TOPCONS2 also provides two quantitative profiles: (i) a per-residue reliability score, derived from the agreement among predictors, and (ii) a ΔG insertion energy profile, estimating the free energy cost (in kcal mol⁻¹) of inserting each residue into the membrane based on an experimentally derived biological hydrophobicity scale. Graphical outputs include individual prediction tracks for each integrated algorithm, a consensus topology map, and two continuous plots for the reliability and ΔG insertion profiles.\u003c/p\u003e\u003cp\u003eFor structural modeling, AlphaFold2 was executed via the ColabFold platform (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://colabfold.mmseqs.com\u003c/span\u003e\u003cspan address=\"https://colabfold.mmseqs.com\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e)\u003c/span\u003e, using MMseqs2-based homology detection and AlphaFold’s Evoformer network to generate high-accuracy three-dimensional structures. To characterize potential pore formation, the AlphaFold2 model was submitted to PoreWalker (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ebi.ac.uk/thornton-srv/software/PoreWalker/\u003c/span\u003e\u003cspan address=\"https://www.ebi.ac.uk/thornton-srv/software/PoreWalker/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e)\u003c/span\u003e, a cavity and channel analysis algorithm that identifies transmembrane pores, computes diameter profiles at 1 Å resolution, classifies geometric segments (SDUS: Straight–Diverging–Uniform–Straight), and maps pore-lining residues.\u003c/p\u003e\u003cp\u003eLigand-binding site predictions were performed using Prank2 (PrankWeb, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://prankweb.cz/\u003c/span\u003e\u003cspan address=\"https://prankweb.cz/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), a machine learning-based platform developed by Charles University as part of ELIXIR's European bioinformatics infrastructure. The AlphaFold2 structure of ORF1 was analyzed using the platform's default parameters with the original structure configuration. The algorithm identified and ranked potential binding pockets based on volume, surface area, and druggability scores. Protein structure visualization and cavity property mapping were conducted using the integrated PrankWeb visualization tools.\u003c/p\u003e\u003cp\u003eMolecular docking simulations were performed using SwissDock (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.swissdock.ch\u003c/span\u003e\u003cspan address=\"http://www.swissdock.ch\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e)\u003c/span\u003e, where the AlphaFold2 structure of ORF1 was used as a rigid receptor. Ligands included divalent cations Ca²⁺ and Mg²⁺ (modeled as charged spheres), ATP (structure retrieved from PubChem and optimized using Open Babel and Avogadro), and a biosynthetic oligomeric PHB–PHV–phosphate construct, reflecting the spectroscopically characterized polymer [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Binding poses were ranked based on full fitness scores and ΔG binding affinity. Molecular interactions and ligand placements were visualized using UCSF ChimeraX (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.cgl.ucsf.edu/chimerax/\u003c/span\u003e\u003cspan address=\"https://www.cgl.ucsf.edu/chimerax/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e).\u003c/span\u003e\u003c/p\u003e\u003cp\u003eSpectroscopic validation of the polymer’s phosphate and ester functionalities was derived from previously reported FTIR, ¹H NMR, and ¹³C NMR signals [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] consistent with phosphorylated co-polyesters. GC–MS data supported the presence of mono-, di-, and triphosphorylated oligomers aligning with ligand docking models. Phosphatetocarbonyl ratios in spectroscopic data were calculated as band or peak count divided by total bands. Two-dimensional data visualizations were generated using Matplotlib 3.8 in Python 3.11. All graphical outputs maintained the native resolution and quality parameters of the respective software tools.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability:\u0026nbsp;\u003c/strong\u003eAll data generated or analyzed during this study are included in this published article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e: Not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e: The author has read and approved the manuscript and consent to its publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e: The author declares no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e: This research received no external funding.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical trial:\u0026nbsp;\u003c/strong\u003eno clinical trial registration is applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e: Dr. Desouky Abd‑El‑Haleem conceptualized, developing the methodological framework encompassing membrane‑topology prediction, structural modeling, pore analysis and molecular docking. He performed all computational data acquisition and curation, carried out formal data analysis and interpretation, prepared the figures, and drafted the manuscript. He critically revised the text for intellectual content and approved the final version for publication.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eReddy, C. S. K.; Ghai, R.; Rashmi; Kalia, V. C. Polyhydroxyalkanoates: an overview. \u003cem\u003eBioresour. Technol.\u003c/em\u003e\u003cstrong\u003e87\u003c/strong\u003e, 137\u0026ndash;146 (2003). https://doi.org/10.1016/S0960-8524(02)00212-2 \u003c/li\u003e\n\u003cli\u003eChen, G.-Q. A microbial polyhydroxyalkanoates (PHA) based bio‑ and materials industry. \u003cem\u003eChem. Soc. Rev.\u003c/em\u003e\u003cstrong\u003e38\u003c/strong\u003e, 2434\u0026ndash;2446 (2009). https://doi.org/10.1039/B812677C\u003c/li\u003e\n\u003cli\u003eJendrossek, D.; Pfeiffer, D. 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Structure of the mechanosensitive channel of small conductance MscS. \u003cem\u003eNature\u003c/em\u003e\u003cstrong\u003e431\u003c/strong\u003e, 963\u0026ndash;970 (2004). https://doi.org/10.1038/nature03085\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Supplementary Data 1","content":"\u003cp\u003eSupplementary Data 1 is not available with this version\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"ORF1, polyhydroxyalkanoate, polyester channel, membrane topology, AlphaFold2, PoreWalker, molecular docking, spectroscopy","lastPublishedDoi":"10.21203/rs.3.rs-6506981/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6506981/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMicrobial polyhydroxyalkanoates (PHAs) have traditionally been viewed as inert carbon reserves, yet emerging evidence implicates these polyesters in ion transport and stress response. Here, we identify and characterize ORF1 from \u003cem\u003eHanseniaspora valbyensis\u003c/em\u003e as the first genetically encoded polyester synthase\u0026ndash;translocator that couples phosphorylated poly[(R)-3-hydroxybutyrate\u0026ndash;3-hydroxyvalerate] (PHB\u0026ndash;PHV) biosynthesis with vectorial export through a membrane pore. The 344residue ORF1 protein was heterologously expressed in \u003cem\u003eEscherichia coli\u003c/em\u003e ΔphaC, and its product validated by TEM, FTIR, NMR and GC\u0026ndash;MS. Six independent topology predictors (Phobius, PolyPhobius, MEMSATSVM, Philius, DeepTMHMM and MemBrain) and a TOPCONS2 metaanalysis converged on a central multihelix region (residues 159\u0026ndash;222) as the membraneembedded core. AlphaFold2 modeling and PoreWalker analysis revealed an amphipathic channel with alternating hydrophobic clamps and electrostatic constrictions (SDUS geometry). PrankWeb pocket mapping and SwissDock simulations demonstrated that divalent cations (Ca\u0026sup2;⁺, Mg\u0026sup2;⁺), ATP and phosphorylated PHB\u0026ndash;PHV oligomers occupy overlapping binding corridors, stabilized by aromatic, aliphatic and basic residues. Spectroscopic signatures of phosphate incorporation and aliphatic backbone structure corroborated in silico interaction models. This bifunctional architecture elevates PHAs from metabolic stores to active mediators of membrane homeostasis and stress adaptation. ORF1 defines a new class of polyesterbased channels, unifying biopolymer synthesis and transport within a single molecular scaffold, and offers a platform for engineering bespoke polymer conduits in synthetic biology.\u003c/p\u003e","manuscriptTitle":"Bifunctional Polyester Synthase–Channel Driving Phosphorylated PHB–PHV Synthesis and Ion Conductance","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-13 18:43:20","doi":"10.21203/rs.3.rs-6506981/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"186e64f6-9ab2-4729-89ff-b4589786c389","owner":[],"postedDate":"May 13th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-06-17T08:39:30+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-13 18:43:20","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6506981","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6506981","identity":"rs-6506981","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}