Structural and Functional Insights into a Previously Uncharacterized Psr LCP Protein from Streptococcus dysgalactiae subsp. dysgalactiae

Structural and Functional Insights into a Previously Uncharacterized Psr LCP Protein from Streptococcus dysgalactiae subsp. dysgalactiae | 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 Structural and Functional Insights into a Previously Uncharacterized Psr LCP Protein from Streptococcus dysgalactiae subsp. dysgalactiae João Paquete-Ferreira, Leonor Torres, Joana Bryton, Marino F. A. Santos, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8368946/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 There is a pressing need to develop new antibiotics targeting alternative biological pathways to effectively tackle the problem of antibiotic resistance. LytR-CpsA-Psr (LCP) proteins catalyze the attachment of cell-wall glycopolymers to peptidoglycan, a crucial step in Gram-positive bacterial envelope biogenesis and a promising target for antimicrobial development. Here we report the structural and functional characterization of a Psr-type LCP protein from Streptococcus dysgalactiae subsp. dysgalactiae (SDSD). Phylogenetic analysis places this enzyme within the Psr/LcpA clade, showing higher conservation among streptococcal homologs than with LytR-type enzymes. The AlphaFold2 model and small-angle X-ray scattering (SAXS) data reveal a conserved overall fold with subtle differences from LytR, including an elongated shape and flexible termini. Sequence analysis highlights distinctive Psr-specific residues (R235, F317, F320) that may influence substrate recognition. Enzymatic activity was confirmed using both colorimetric and nuclear magnetic resonance (NMR) assays, demonstrating conversion of ADP to AMP with maximal activity near 37°C and an apparent zero-order kinetic regime under saturating substrate conditions. The ¹H NMR assay, implemented here for the first time, provides a continuous, label-free approach to monitor LCP activity in real time. Together, these findings establish the structural conservation and functional distinctiveness of the SDSD Psr LCP domain, introduce a complementary NMR-based strategy suitable for kinetic and inhibitor studies within the LCP family and lay the groundwork for a deeper understanding of LCP-mediated processes, supporting their potential as antibiotic targets. Psr LCP protein Streptococcus dysgalactiae Antibiotic resistance Small-angle X-ray scattering Nuclear Magnetic Resonance Protein structure and function Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Key points • An unannotated SDSD protein is identified as Psr LCP by phylogenetics. • Structural analyses reveal a conserved fold with Psr-specific features. • A new ¹H NMR assay enables real-time, label-free LCP kinetics. Introduction The rise of antibiotic-resistant bacteria is one of the most pressing global health threats of our time (Frieri et al. 2017 ; Miethke et al. 2021; Salam et al. 2023 ; Sengupta et al. 2013 ; Ventola 2015 ). The emergence of multidrug-resistant strains such as methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococci (VRE), and carbapenem-resistant Enterobacteriaceae (CRE) has rendered many antibiotics ineffective (Frieri et al. 2017 ), underscoring the need to identify new therapeutic targets within essential bacterial pathways. In Gram-positive bacteria, cell-wall integrity and remodeling rely on the coordinated action of enzymes that synthesize, modify, and anchor glycopolymers to peptidoglycan (Do et al. 2020 ; Rausch et al. 2019 ). Among these, the LytR-CpsA-Psr (LCP) family, present in virtually all Gram-positive species, catalyzes the final transfer of wall teichoic acids (WTAs) and other cell-wall glycopolymers (CWGPs) onto the peptidoglycan backbone, a reaction essential for cell-wall assembly, division, and biofilm formation (Gale et al. 2017 ; Harrison et al. 2016 ; Hübscher et al. 2008 ; Kawai et al. 2011 ; Stefanović et al. 2021 ). Because this process occurs on the outer surface of the cell membrane, LCP enzymes are accessible to inhibitors and have emerged as promising antimicrobial targets (Kawai et al. 2011 ; Stefanović et al. 2021 ). Yet, despite their physiological importance and widespread conservation, detailed structural and functional information for these proteins remains limited. During biosynthesis, CWGPs are linked to an undecaprenyl carrier via a pyrophosphate bond (Fig. 1 ). LCP proteins act as pyrophosphatases, cleaving this bond and transferring the CWGP from the lipid carrier to the peptidoglycan, forming a new phosphodiester linkage (Gale et al. 2017 ; Harrison et al. 2016 ; Hübscher et al. 2008 ; Kawai et al. 2011 ; Stefanović et al. 2021 ). Despite their variable topology and occasional accessory domains, and even with low sequence identity among homologs (including those within the same species) LCP proteins share a highly conserved overall fold. This includes a hydrophobic pocket that accommodates the lipid carrier and coordinates a divalent cation, typically Mg²⁺, together with a pyrophosphate-containing substrate (Hübscher et al. 2008 ; Kawai et al. 2011 ; Stefanović et al. 2021 ). Although the pocket itself is universal, its residue composition tend to be conserved only within a given subfamily of proteins. Several subfamilies (LytR/BrpA, CpsA, Psr/LcpA, LcpB/TagT, LcpC/TagU and LcpD/TagV) are commonly found across Gram-positive species, often coexisting within the same genome (Kawai et al. 2011 ; Schaefer et al. 2017 ; Zilla et al. 2015b ). Structural and biochemical data are available for members of the different subfamilies (Kawai et al. 2011 ; Schaefer et al. 2018 ; Siegel et al. 2019 ); however, only the LcpA/Psr protein from S. aureus has been structurally characterized (Li et al. 2020 ), while the corresponding proteins from streptococcal species remain poorly characterized. Crystal structures of LCP proteins with bound analogues of the lipidic carrier containing phosphate or diphosphate moieties reveal the charged portion of the substrate positioned near the pocket entrance, where catalysis occurs (Eberhardt et al. 2012 ; Kawai et al. 2011 ; Li et al. 2020 ; Schaefer et al. 2018 ). Four conserved arginine residues stabilize the negatively charged diphosphate group (Fig. 2 ), and their mutation (e.g., R118A, R219A, R227A in Bacillus subtilis TagT) abolishes activity (Schaefer et al. 2018 ). Two conserved aspartates coordinate the Mg²⁺ cofactor, which adopts an octahedral geometry completed by two water molecules and two substrate oxygens (Fig. 2 ) (Kawai et al. 2011 ; Schaefer et al. 2018 ). The metal ion is essential for catalysis, as activity decreases sharply in the presence of chelators such as EDTA or upon mutation of either aspartate (Gale et al. 2017 ; Kawai et al. 2011 ). Streptococcus dysgalactiae subsp. dysgalactiae (SDSD) is an opportunistic pathogen associated with bovine mastitis, fish streptococcosis, and, increasingly, human infections. Its genome encodes multiple LCP homologs, suggesting functional diversification related to cell-envelope remodeling and virulence. Because these enzymes operate extracellularly and catalyze an essential step in cell-wall assembly, they are considered promising antibiotic targets (Hübscher et al. 2008 ; Sewell and Brown 2014 ). However, inhibition is complicated by the frequent presence of several partially redundant LCP genes within a single organism (Over et al. 2011 ). In many bacteria, single knockouts have only mild effects on growth or virulence, whereas multiple deletions lead to severe envelope defects or lethality (Dengler et al. 2012 ; Over et al. 2011 ). Understanding the specific structural and functional traits that distinguish individual LCP proteins is therefore critical for designing selective or synergistic inhibitors. Here, we report the structural and functional characterization of two LCP proteins from S. dysgalactiae subsp. dysgalactiae : LytR (previously described in (Paquete-Ferreira et al. 2024 )) and a newly characterized Psr-type enzyme. Using phylogenetic analysis, AlphaFold2 modeling, and small-angle X-ray scattering (SAXS), we show that this enzyme adopts the canonical LCP fold but exhibits distinct structural features relative to LytR. Sequence analysis highlights residues potentially associated with Psr-specific substrate recognition. Enzymatic activity was confirmed by colorimetric and nuclear magnetic resonance (NMR) assays, demonstrating ADP-to-AMP conversion and metal dependence. Importantly, the continuous, label-free, ¹H NMR approach introduced here provides a complementary tool for assessing LCP activity in real time and sets the stage for future kinetic and inhibitor studies. Methods Genetic construction and site-directed mutagenesis The construct for the LCP domain of the Psr protein from Streptococcus dysgalactiae subsp. dysgalactiae (SDSD) was produced by amplifying the gene fragment encoding residues 213–480, corresponding to the LCP domain, from genomic DNA (VSD9 isolate). The amplified fragment was cloned into the pNIC28-Bsa4 vector, which encodes an N-terminal hexahistidine tag followed by a TEV protease cleavage site, using the ligase-independent cloning (LIC) system. The primers used are listed in Table S1 (Supplementary Material). The construct excludes the membrane-anchoring helix located between the two domains to ensure soluble expression of the catalytic LCP region. The production of the LytR LCP domain construct was performed as previously described (Paquete-Ferreira et al. 2024). Protein expression and purification For the production of the LCP domain of Psr, the construct was transformed into E. coli BL21 (DE3) cells. Cultures were grown in LB medium supplemented with kanamycin (50 µg/mL) at 37 °C and 180 rpm. When the optical density (OD₆₀₀) reached 0.5–0.8, protein expression was induced with 1 mM IPTG, and incubation continued overnight at 16 °C and 180 rpm. Cells were harvested by centrifugation (8,000 rpm, 10 min, 4 °C), resuspended in lysis buffer (10 mM K₂HPO₄ pH 6.0, 500 mM NaCl, 5 mM MgCl₂, and 10 mM imidazole), and disrupted by sonication (10 s on, 20 s off, 50 % amplitude) for 10 min. The lysate was clarified by centrifugation (11,000 × g, 30 min, 6 °C), and the supernatant was loaded onto a 5 mL Ni²⁺ HisTrap™ HP column (Cytiva) for immobilized metal-affinity chromatography (IMAC). The protein was eluted using a linear imidazole gradient (30–500 mM). Fractions containing the Psr LCP domain were dialyzed overnight at 4 °C against 1 L of 10 mM sodium citrate pH 6.0, 500 mM NaCl, and 5 mM MgCl₂ with gentle stirring. The dialyzed protein was concentrated and further purified by size-exclusion chromatography (SEC) using a Superdex 200 Increase 10/300 GL column (Cytiva). Fractions containing the monomeric protein were pooled, concentrated to 10 mg/mL, and stored at −20 °C. The LytR LCP domain was expressed and purified following the protocol previously described (Paquete-Ferreira et al. 2024). Small-angle X-ray scattering (SAXS) Samples of the LytR and Psr LCP domains were prepared at concentrations of 7.7 mg/mL and 4.2 mg/mL, respectively, in 50 mM HEPES pH 8.0, 150 mM NaCl, and 5 mM MgCl₂, and subsequently subjected to five serial dilutions. SAXS experiments were conducted at the European Synchrotron Radiation Facility (ESRF, Grenoble, France) on beamline BM29. Data inspection, reduction, and preliminary analysis were performed using PRIMUS (Konarev et al. 2003), allowing determination of the radius of gyration (R g ) and forward scattering intensity (I₀) from Guinier plots and pair-distance distribution functions. A summary of data collection and analysis parameters is provided in Table 1. Theoretical scattering curves from the crystallographic and modeled structures were calculated and compared to the experimental profiles using Crysol and SREFLEX (Svergun et al. 1995). Ab initio shape reconstructions were generated with DAMMIF, and the resulting models were averaged and superimposed using DAMAVER (Franke and Svergun 2009; Panjkovich and Svergun 2016). Table 1 : SAXS samples and analysis a. Sample details Organism Streptococcus dysgalactiae subsp. dysgalactiae Source E. coli (BL21) recombinant expression Scattering particle composition LytR Psr Protein LytR LCP domain Psr LCP domain Ligand - Stoichiometry of components - Solvent composition 50 mM HEPES pH 8.0, 150 mM NaCl and 5 mM MgCl 2 Sample concentration (mg/ mL) 7.7 4.2 b. SAS data collection ESRF BM29 Data-acquisition/reduction software BSXCuBE Source/instrument description 2 Pole Wiggler, Pilatus3 2M in-vacuo Measured q-range (q min - q max ) (Å -1 ) 0.0042 – 0.522 Exposure time (s), No. of exposures 10 frames c. SAS-derived structural parameters Method(s)/software LytR Psr Guinier analysis I(0) ± 𝜎 (cm -1 ) 14.86 ± 0.079 9.58 ± 0.083 Rg ± 𝜎(nm) 2.50 ± 0.02 2.48 ± 0.03 qRg range (datapoint range) 0.41-1.30 (24-93) 0.41-1.29 (25-93) Linear fit assessment (AUTORG fidelity) 0.66 0.45 PDDF/P(r) analysis I(0) ± 𝜎 (cm -1 ) 15.01 ± 0.063 9.58 ± 0.077 Rg ± 𝜎(nm) 2.62 ± 0.011 2.53 ± 0.028 dmax (nm) 8.87 9.28 q-range (nm -1 ) 0.16-3.20 0.16-3.25 p(r) reciprocal-space fit 0.83 0.78 Malachite Green Assay The malachite green assay was used to monitor the pyrophosphatase activity of the Psr LCP domain using ADP as substrate, following the procedure previously described (Paquete-Ferreira et al. 2024). To determine the optimal temperature for enzymatic activity, the wild-type Psr LCP domain (42 µM) was incubated with ADP (750 µM) in 50 mM HEPES pH 8.0, 150 mM NaCl, and 5 mM MgCl₂. Reactions were carried out overnight at 4, 20, 37, 42, and 50 °C. Nuclear Magnetic Resonance (NMR) spectroscopy The enzymatic activity of the Psr LCP domain was also monitored by ¹H NMR spectroscopy using ADP as substrate. The reaction was followed by acquiring a series of 1,500 consecutive ¹H spectra (32 s each; total acquisition time ≈13.3 h), measuring the intensity of the peaks corresponding to the H8 proton of the purine moiety of both ADP and AMP. The reaction mixture contained 75 µM protein and 1.5 mM ADP in 10 mM sodium citrate pH 6.0, 500 mM NaCl, 5 mM MgCl₂, 10 % D₂O, and 50 µM 4,4-dimethyl-4-silapentanesulfonic acid (DSS) as an internal chemical-shift reference. After preparation, the sample was transferred to a 3 mm NMR tube and immediately placed in the spectrometer; acquisition started approximately 255 s later. A control sample containing ADP without protein was analyzed under identical conditions. NMR spectra were recorded at 293 K on a 600 MHz Bruker Avance III spectrometer (proton Larmor frequency 600.13 MHz) equipped with a 5 mm TCI cryoprobe. Data were processed using TopSpin 4.2.0 (Bruker BioSpin). All ¹H spectra were acquired with a spectral width of 9615.39 Hz centered at 2806.21 Hz, using eight transients, 32k data points, and a relaxation delay of 1.0 s. Solvent suppression was achieved by excitation sculpting with gradients (Hwang and Shaka 1995), applying a selective pulse (Squa100.1000) of 2 ms duration to irradiate the solvent signal. Results SDSD Psr clusters within the Psr/LcpA subfamily A common feature of the LCP protein family is the presence of multiple genes encoding this domain. In SDSD, there are two: the previously described LytR/BrpA (Paquete-Ferreira et al. 2024 ) and a second, uncharacterized protein analyzed here (Psr). The two share low overall sequence identity (12.9%), with the LCP domains showing 21.9% identity, a divergence similar to that observed among LCP paralogs in other species such as Bacillus subtilis , Streptococcus pneumoniae , and Staphylococcus aureus . The proteins also differ topologically: while LytR/BrpA includes a membrane-anchoring helix and two extracellular domains (the LCP core and a C-terminal accessory domain), Psr contains an intracellular N-terminal region, a transmembrane helix, and a single extracellular LCP domain. To examine the evolutionary relationship of SDSD Psr within the LytR-CpsA-Psr (LCP) family, its sequence was aligned with representative LCP genes from B. subtilis , (proteins categorized as TagTUV), S. aureus , Bacillus anthracis (proteins categorized as LcpA, LcpB, LcpC and LcpD), S. pneumoniae , S. agalactiae and S. mutans (proteins categorized as LytR/BrpA, CpsA and Psr). Multiple sequence alignment (ClustaL Omega (Sievers and Higgins 2017)) indicated that SDSD Psr retains the conserved motifs characteristic of LCP enzymes, consistent with the presence of key catalytic residues. Phylogenetic analysis performed using IQ-TREE web server with 1,000 ultrafast bootstraps (Trifinopoulos et al. 2016 ), placed SDSD Psr within the Psr/LcpA subfamily, clustering closely with other streptococcal homologs and clearly separating from LytR-type enzymes (Fig. 3 ). These results agree with those by L. Zilla and colleagues (Zilla et al. 2015a ). Specifically, B. subtilis TagT clusters with S. aureus and B. anthracis LcpB proteins, while B. subtilis TagU is in the same branch as S. aureus and B. anthracis LcpC proteins. Additionally, B. subtilis TagV clusters with B. anthracis LcpD, and S. aureus LcpA is found on a separate branch. By contrast, proteins characterized as LytR/BrpA and CpsA form branches distinct from the LcpB/C/D clades. The uncharacterized SDSD protein, as well as all proteins categorized as Psr, are clustered within the same branch as S. aureus LcpA. This observation aligns with existing NCBI annotations, where S. aureus LcpA is described as MsrR - a designation similar to S. mutans Psr. Together with the observed topology and the fact that S. aureus LcpA can also be categorized as a Psr protein, this supports its classification as a Psr-type protein and suggests functional similarity to LcpA-like enzymes described in Staphylococcus aureus and Bacillus subtilis . This classification is consistent with phylogenetic evidence and reduces potential confusion. Additionally, our analysis identified S. agalactiae CAD46000 as a LytR/BrpA protein, contributing to a clearer understanding of LCP protein classification within these species. According to this analysis, all Streptococcus species contain a LytR protein. Notably, S. agalactiae is the only Streptococcus species that lacks a Psr protein from the group under study. Also, along with S. pneumoniae , this is one of the only two species encoding CpsA proteins. Therefore, the data suggest that in the Streptococcus genus, LytR is likely essential, Psr may serve a comparable role, and CpsA appears to have a more specialized function, consistent with its known involvement in capsule synthesis and transfer. Collectively, these findings indicate that Psr proteins diverged early from the rest of the LCP family, reflecting an ancient evolutionary split. Sequence similarities are stronger among functional subtypes than among species, implying that each LCP class has been shaped by similar selective pressures. This observation supports the hypothesis that horizontal gene transfer has contributed to the distribution and diversification of LCP enzymes. Predicted structure shows a conserved LCP fold To predict the 3D structure of the designed SDSD Psr construct and compare it with related proteins, we modeled the Psr LCP domains from Streptococcus pyogenes , S. mutans , and S. pneumoniae identified in the phylogenetic analysis using AlphaFold 2 (AF2) (Jumper et al. 2021 ) (Fig. 4 ). All structures yielded high-confidence predictions for the LCP domain with a per-residue confidence (pLDDT) > 90 for most of the core region (Figure S1 ). The SDSD Psr model displays the canonical LCP architecture, consisting of a mixed α/β-fold in which a central β-sheet is surrounded by α-helices forming a compact catalytic core. The predicted topology includes a single transmembrane helix that connects to the LCP domain by a flexible region, consistent with other LCP enzymes (Stefanović et al. 2021 ). Sequence identities and RMSD values of the Psr LCP domains (including LytR) relative to the SDSD Psr LCP domain, as well as hydrophobic-pocket volumes calculated with CASTp (Tian et al. 2018 ), are summarized in Table 2 . Table 2 Comparison of sequence identity, RMSD values and the pocket volume of SDSD Psr with the other identified Psr proteins and SDSD LytR Protein Sequence identity relative to SDSD Psr (%) RMSD relative to SDSD Psr (aligned Cα) Pocket volume (Å 3 ) SDSD Psr (AF2 model) - - 652 SDSD LytR (PDB ID: 8QTY)(Paquete-Ferreira et al. 2024 ) 24.2 1.72 Å (150/150) 411 S. pyogenes Psr (AF2 model) 75.0 0.39 Å (211/211) 561 S. pneumoniae Psr (AF2 model) 54.2 0.55 Å (202/202) 521 S. mutans Psr (AF2 model) 63.5 0.47 Å (210/210) 609 S. aureus LcpA (PDB ID: 6UEX)(Li et al. 2020 ) 32.2 1.09 Å (174/174) 549 Analysis of these data shows that the streptococcal Psr LCP domains share higher sequence identity (54.2–75.0%) with SDSD Psr than either SDSD LytR (24.2%) or S. aureus LcpA (32.2%), a trend also reflected in their lower RMSD values. These results reinforce the phylogenetic grouping described above. Pocket-volume measurements do not reveal a consistent pattern, displaying variable values even among the streptococcal Psr LCP domains. Given the high similarity among Psr LCP domains, we performed a multiple-sequence alignment followed by structural comparison to evaluate whether the variability observed across the LCP family is reduced within this subgroup. The alignment included all Psr LCP domains and SDSD LytR, while the structural analysis used the SDSD Psr AF2 model, the SDSD LytR crystal structure (PDB ID: 8QTY (Paquete-Ferreira et al. 2024 )), and the S. aureus LcpA structure (PDB ID: 6UEX (Li et al. 2020 )) (Fig. 4 ). Although residue variability is distributed throughout the structure, we focused on differences at the peptidoglycan-binding site and within the hydrophobic pocket, where some hydrophobic residues, despite maintaining similar chemical properties, are not fully conserved. Within the peptidoglycan-binding site, residue R235 (SDSD Psr numbering) appears to be unique to Psr proteins, being replaced by an asparagine in SDSD LytR. In contrast, residues K278 and D392 are more broadly conserved between both LCP sub-types. Similarly, in the hydrophobic pocket, residues F317 and F320 are conserved among Psr proteins but replaced by methionine and leucine, respectively, in SDSD LytR, whereas L380 remains unchanged. The sequence alignment (Figure S2) also revealed a motif distinguishing LytR and Psr proteins: LytR enzymes contain RMR(X)₆DXGR, whereas Psr proteins exhibit RFR(X)₅DXGR. The substitution of methionine for phenylalanine and the one-residue shorter spacing likely influence substrate binding, suggesting distinct preferences for peptidoglycan, at different maturation stages, between the two LCP subtypes. Psr displays the canonical LCP architecture in solution To experimentally validate the AF2 model and compare it with LytR, we performed small-angle X-ray scattering (SAXS) analysis. Initial data reduction and preliminary analysis revealed concentration-dependent effects in the scattering profiles, reflected in variations of the radius of gyration (R g ). Nevertheless, R g and forward scattering intensity (I₀) values could be reliably extracted for both proteins (Table 2 ). To determine the pair-distance distribution functions p(r), selected curves were merged by combining low-angle data from the lowest concentration with high-angle data from the highest concentration. This approach yielded D max values. Figure 5 shows the merged scattering curves for LytR and Psr (A) and their corresponding p(r) functions (B). The comparison of the SAXS profiles indicates that the two proteins display broadly similar scattering behavior, with a χ² value of 2.40. The main differences occur at higher angles, suggesting an overall comparable architecture with local structural variations. These may arise from the His-tags being positioned at opposite termini: C-terminal in LytR and N-terminal in Psr. The p(r) functions further suggest a slightly more elongated structure for Psr and a more flexible conformation for LytR. Following the methodology previously described (Paquete-Ferreira et al. 2024 ), the experimental SAXS data were compared with theoretical scattering curves generated from the LytR crystal structure (PDB ID: 8QTY (Paquete-Ferreira et al. 2024 ); missing regions modelled with MODELLER) and the Psr AF2 model. The theoretical fits showed moderate agreement (χ² = 2.82 for LytR and 2.36 for Psr). Refinement with SREFLEX improved the fits (χ² = 2.44 and 2.08, respectively) and yielded RMSD values of 5.07 Å for LytR and 5.92 Å for Psr. In both cases, the global fold was preserved in solution, with the largest deviations occurring at the N-terminus of Psr and the C-terminus of LytR. Ab-initio envelopes were generated for both proteins, yielding χ² values of 0.98 for Psr and 1.13 for LytR, indicating excellent agreement between data and models. Figure 6 shows the averaged DAMFILT envelopes produced with DAMAVER, the corresponding best DAMMIF models, and their superpositions with the respective structures, calculated using CIFSUP. Both Psr and LytR fit well within their SAXS envelopes, occupying most of the reconstructed volume. This correspondence indicates minimal conformational differences between the solution and modeled structures, confirming their structural stability in solution. Nevertheless, subtle differences distinguish the two proteins. The Psr envelope exhibits a shallow cleft that may correspond to a flexible or interaction region, whereas the LytR envelope appears more compact and uniform, suggesting a more rigid structure. Additionally, one loop near the cleft in the Psr model adopts a different conformation from that in LytR, potentially contributing to its greater flexibility or accessibility. Neither protein displays a strictly globular shape; both include an additional, smaller region that imparts asymmetry, though its position differs between the two. These observations are consistent with our previous results (Paquete-Ferreira et al. 2024 ), supporting the view that Psr and LytR share a similar overall conformation, with the main differences localized to mobile or flexible regions. Functional characterization of the Psr LCP domain To assess the enzymatic activity of the SDSD Psr LCP domain, we monitored the conversion of ADP into AMP using both the malachite green assay and ¹H NMR spectroscopy. In our previous work on the SDSD LytR domain (Paquete-Ferreira et al. 2024 ), ADP was introduced as a soluble substrate for evaluating LCP pyrophosphatase activity; here, we extend that concept by using ¹H NMR to monitor the reaction directly in solution. Temperature dependence The optimal temperature for Psr activity was determined by malachite green assay at five temperatures (4, 20, 37, 42, and 50°C). The reactions were incubated overnight, with samples taken at the start and end points (Fig. 7 A). The enzyme displayed maximal activity at 37°C, achieving 77.2% conversion. Notably, Psr remained active up to 50°C, with conversions of 18.4% (4°C), 34.1% (20°C), 49.2% (42°C), and 49.0% (50°C). These results indicate good thermal stability across a range of temperatures and a clear preference for higher temperatures. NMR assay for real-time monitoring To follow the reaction continuously, we implemented a label-free ¹H NMR assay. Comparison of the 1D spectra of ADP and AMP (Figure S3) revealed distinct chemical shifts for the H8 proton of the purine moiety, enabling real-time tracking of substrate consumption and product formation (Fig. 7 B). Notably, the ¹H NMR spectra already exhibited a small AMP signal at the start of the reaction, attributed to trace AMP present in the ADP reagent. This baseline signal was constant across all samples. During the enzymatic reaction, the AMP peak increased steadily, reflecting conversion of ADP to AMP. Control experiments confirmed that, in the absence of enzyme, the AMP signal did not change (Figure S3), demonstrating that ADP does not undergo spontaneous hydrolysis under the assay conditions. Although the optimal activity was observed at 37°C, NMR experiments were conducted at 20°C to prevent protein precipitation inside the NMR tube. Under these conditions, ~ 25% conversion of ADP to AMP was achieved. Kinetic behavior and inhibition Continuous monitoring by ¹H NMR showed a linear evolution of ADP and AMP signals over time suggesting that, under the experimental conditions used, the reaction proceeds in an apparent zero-order regime. This steady-state behaviour is consistent with substrate saturation and demonstrates that the assay quantitatively captures catalytic progression. To validate the assay and assess inhibition, two negative controls were performed: (i) in the absence of enzyme and (ii) in the presence of enzyme with EDTA (Figure S3). Although EDTA did not fully inhibit activity, a clear reduction was observed, consistent with Mg²⁺ chelation and the reported metal dependence of LCP enzymes (Kawai et al. 2011 ). We note that the use of a citrate buffer at pH 6.0 with 5 mM MgCl₂ likely reduced the effective chelating capacity of EDTA; however, this limitation does not affect the interpretation of the observed partial inhibition, which remains fully consistent with a Mg²⁺-dependent catalytic mechanism. Discussion This work combines phylogenetic, structural, and biochemical analyses to characterize a Psr-type LCP protein from Streptococcus dysgalactiae subsp. dysgalactiae (SDSD). By integrating computational modeling, SAXS, and real-time NMR assays, we provide new insights into the structural conservation, catalytic properties, and metal dependence of this enzyme. The results highlight both the shared and distinctive features of Psr relative to other LCP subfamilies and establish a methodological basis for quantitative, label-free monitoring of LCP activity. The phylogenetic analysis of the LCP family provides valuable insights into the classification and functional diversity of these enzymes. Our results support the division of the family into six sub-types: LytR/BrpA, CpsA, Psr/LcpA, LcpB, LcpC, and LcpD. These sub-types form well-defined and independently supported branches in the phylogenetic tree, with additional members clustering consistently within each subtype. This organization offers a practical framework for classifying uncharacterized LCP proteins, complementing existing nomenclature and facilitating the identification of new sub-families that expand our understanding of LCP functional diversity. Within this framework, LytR, LcpB, LcpC, and LcpD proteins appear more closely related to each other, whereas CpsA and especially Psr show greater divergence from the rest of the family. The early divergence of the Psr subgroup suggests unique evolutionary pressures or functional adaptations, possibly related to differences in the glycopolymers transferred to the peptidoglycan. Further structural and biochemical studies are required to determine whether this divergence reflects substrate specificity or variations in catalytic function. The SAXS data indicate that the LCP domains of LytR and Psr share an overall similar structure, with no evidence of significant disorder in either protein. This indicates that identical buffer conditions did not affect their global architecture, despite the different theoretical pI values of LytR (6.25) and Psr (5.02). The slightly higher D max observed for Psr points to a more elongated structure, possibly due to increased flexibility of the His-tag at its N-terminus compared with the C-terminal tag of LytR. The use of ¹H NMR to monitor nucleotide conversions, particularly the hydrolysis of ATP to ADP, is well established (Lian et al. 2016 ). Similar to earlier ATPase studies, the reaction was monitored through the distinct chemical shifts of the H8 proton of the purine ring (Guo et al. 2014 ). A key advantage of ¹H NMR over the malachite-green assay is its ability to collect multiple time points from the same sample throughout the reaction, eliminating the need for larger reaction volumes. Moreover, ¹H NMR allows simultaneous observation of substrate depletion and product formation, providing a more comprehensive view of reaction progress. Both techniques yielded comparable conversions, with differences explained by reaction time and buffer conditions. At 20°C, the malachite-green assay yielded 34.1 % conversion, while ¹H NMR showed 5.4 %. This difference can largely be ttributed to the assay duration (≈ 18 h versus 13 h). Assuming the apparent zero-order kinetics observed, a 5-h extension could account for an additional ~ 9 % conversion, consistent with the iscrepancy. Interestingly, when applying these kinetics to estimate reaction velocities, ¹H NMR yields 0.49 µM min⁻¹ and the malachite-green assay 0.24 µM min⁻¹. The difference likely reflects the lower substrate excess (18-fold versus 20-fold) and the distinct pH conditions used in the two assays (pH 6.0 and 8.0, respectively). In a study by Schaefer and colleagues (Schaefer et al. 2018 ), the enzymatic activity of B. subtilis TagT was also analyzed assuming a zero-order kinetic regime, in line with our findings. Nevertheless, the reaction investigated by Schaefer and colleagues involved glycopolymer transfer, whereas the present work focuses on ADP hydrolysis. Additional studies at lower substrate concentrations will be required to confirm whether the apparent zero-order behaviour reflects enzyme saturation in the ADP to AMP reaction or is a general kinetic feature of the LCP family. For the transfer reaction itself, continuous assays similar to the present one will help clarify whether it also proceeds at a constant rate. The EDTA inhibition assay showed only partial activity reduction. This outcome is consistent with previous reports (Kawai et al. 2011 ) but may also result from the buffer composition used here (10 mM sodium citrate pH 6.0, 500 mM NaCl, 1 mM EDTA). The relatively acidic pH likely decrease the effective chelating capacity of EDTA, allowing residual enzyme activity. Importantly, this limitation does not alter the interpretation of the results, which remain fully consistent with a Mg²⁺-dependent catalytic mechanism. Overall, the combined data demonstrate that the SDSD Psr protein adopts a conserved LCP fold, is catalytically active in vitro , and exhibits robust thermal stability and metal dependence. The continuous ¹H NMR assay introduced here provides a complementary, label-free approach for quantifying LCP activity in real time, paving the way for future kinetic and inhibition studies within this enzyme family. Declarations Acknowledgements The authors would like to thank the staff of beamline BM29 (ESRF) for their assistance during the SAXS data collection. Author Contributions João Paquete-Ferreira: Investigation; methodology; analysis; writing – original draft; writing, review and editing Leonor Torres: Investigation; methodology Joana Bryton: Investigation; methodology Marino F. A. Santos: Methodology; analysis; writing, review and editing Márcia A. S. Correia: Methodology; analysis; writing, review and editing Alexandra R. Fernandes: Supervision; resources; project administration; writing, review and editing Maria João Romão: Supervision; resources; project administration; writing, review and editing Aldino Viegas: Investigation; methodology; analysis; writing, review and editing Teresa Santos-Silva: Conceptualization; supervision; resources; project administration; analysis; writing, review and editing Funding This work is financed by national funds from FCT - Fundação para a Ciência e a Tecnologia, I.P., in the scope of the project UIDP/04378/2020 (DOI: 10.54499/UIDP/04378/2020) and UIDB/04378/2020 (DOI: 10.54499/UIDB/04378/2020) of the Research Unit on Applied Molecular Biosciences - UCIBIO and the project LA/P/0140/2020 (DOI: 10.54499/LA/P/0140/2020) of the Associate Laboratory Institute for Health and Bioeconomy - i4HB., through grant 2020.08580.BD (to J.P.F.), and contracts 2020.00043.CEECIND and 2023.11076TENURE.002 (to A.V.). The NMR spectrometer at NOVA-FCT is part of the National NMR Network (PTNMR) and is supported by national funds from FCT-MCTES through the scope of projects UIDB/04378/ 2020 of the Research Unit on Applied Molecular Biosciences (UCIBIO) and the project UID/50006/2023 of the Associate Laboratory for Green Chemistry (LAQV-REQUIMTE). Data availability The data that support the findings of this study are available from the corresponding author upon reasonable request. Acknowledgements The authors would like to thank the staff of beamline BM29 (ESRF) for their assistance during the SAXS data collection. Ethics approval This study did not involve human participants or animals; therefore, ethical approval was not required. Conflict of interest All authors certify that they have no affiliations with or involvement in any organization or entity with any financial interest or non-financial interest in the subject matter or materials discussed in this manuscript. 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J Bacteriol 197(23):3731–3741 Additional Declarations No competing interests reported. Supplementary Files Supplementarymaterial.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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09:42:17","extension":"png","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":76029,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8368946/v1/9f4622d82ec7184eddf29d11.png"},{"id":98989074,"identity":"d626735a-3344-4d28-b640-32f79122defc","added_by":"auto","created_at":"2025-12-25 09:42:17","extension":"png","order_by":16,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":93133,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8368946/v1/d39c59a0079cda0d177655f2.png"},{"id":99312230,"identity":"e6be14c1-63ce-43dd-930b-d1a3e3358544","added_by":"auto","created_at":"2025-12-31 16:18:21","extension":"png","order_by":17,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":47632,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-8368946/v1/6c5a25745ce3b7e69cfaf555.png"},{"id":98989075,"identity":"dcd9a9a7-4ebb-4330-8ade-eadeccdeba8b","added_by":"auto","created_at":"2025-12-25 09:42:17","extension":"xml","order_by":18,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":112078,"visible":true,"origin":"","legend":"","description":"","filename":"16efa6f97e054b4882c7d3ab33a42b841structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8368946/v1/c442151d35b2bf0670d84fe3.xml"},{"id":98989076,"identity":"e295fa65-5f56-4458-8c0b-26ef0ffde7b4","added_by":"auto","created_at":"2025-12-25 09:42:17","extension":"html","order_by":19,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":124080,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8368946/v1/1c5b8e2e824eb09b6143c179.html"},{"id":98989057,"identity":"31ef98da-2556-497b-8ddc-683c8c4c1225","added_by":"auto","created_at":"2025-12-25 09:42:17","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":963600,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic representation of the intracellular steps in the biosynthesis of wall teichoic acids (WTAs). The glycopolymer is synthesized while attached to an undecaprenyl carrier via a pyrophosphate bond. N-Acetylmannosamine (ManNAc), represented as a green square; N-Acetylglucosamine (GlcNAc) represented as a blue square, and glycerol 3-phosphate represented as a yellow circle.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-8368946/v1/b12c0940b9f63c69659480d2.png"},{"id":99312921,"identity":"e6a34e96-c0a2-4525-8567-68e6f3338301","added_by":"auto","created_at":"2025-12-31 16:19:36","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":912777,"visible":true,"origin":"","legend":"\u003cp\u003eStructural details and proposed catalytic mechanism of Wzg and LCP proteins. Close-up of the \u003cem\u003eS. pneumoniae\u003c/em\u003e Wzg crystal structure active site (PDB ID: 3TEP), showing conserved arginine residues and Mg²⁺-coordinating aspartates as sticks. The Mg²⁺ ion is shown as a green sphere, and the octaprenyl diphosphate as a ball-and-stick model. The coordination sphere includes the two aspartates (sticks), two water molecules (red spheres), and two oxygen atoms from the diphosphate of the octaprenyl diphosphate. The proposed catalytic mechanism for LCP proteins is depicted, highlighting the guanidinium groups of arginine residues R122, R216, and R218 (numbering from \u003cem\u003eS. aureus\u003c/em\u003e LcpA).\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-8368946/v1/ea9726d77e3d95651e80d0cd.png"},{"id":99312362,"identity":"c1a0f34f-d505-48cf-bdbf-b5723dd28a50","added_by":"auto","created_at":"2025-12-31 16:18:53","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":157662,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic relationships among LytR-CpsA-Psr (LCP) proteins from representative Gram-positive bacteria. The tree was constructed from a multiple-sequence alignment of LCP protein sequences from \u003cem\u003eB. subtilis\u003c/em\u003e, \u003cem\u003eS. aureus\u003c/em\u003e, \u003cem\u003eB. anthracis\u003c/em\u003e, \u003cem\u003eS. pneumoniae\u003c/em\u003e, \u003cem\u003eS. agalactiae\u003c/em\u003e, \u003cem\u003eS. mutans\u003c/em\u003e and SDSD. Branch lengths are proportional to evolutionary distance.This figure was generated using the iTOL Interactive Tree of Life online tool.(Letunic and Bork 2024)\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-8368946/v1/c6a6fb9d473e9473bd9d4e4d.png"},{"id":99312856,"identity":"a64d91d0-951b-4cef-822b-873532adc912","added_by":"auto","created_at":"2025-12-31 16:19:32","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2917632,"visible":true,"origin":"","legend":"\u003cp\u003eStructural comparison of LytR-CpsA-Psr (LCP) domains. Superposition of the LCP domains from SDSD Psr (gray, AlphaFold2 model), LytR (black, PDB ID: 8QTY), and \u003cem\u003eS. aureus\u003c/em\u003e LcpA (salmon, PDB ID: 6UEX). Close-up view of the PG-interacting residues, conserved catalytic residues, Mg\u003csup\u003e2+\u003c/sup\u003e coordination residues and hydrophobic residues and, all represented as sticks.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-8368946/v1/8aa816bf52673df589fa9724.png"},{"id":98989068,"identity":"012bfe15-1432-4465-be65-12cdb2a4d0a4","added_by":"auto","created_at":"2025-12-25 09:42:17","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":272751,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of SAXS data from the LytR and Psr proteins. (A) Superposition of the scattering profiles from LytR (black) and Psr (grey). (B) Superposition of their pair-distance distribution functions p(r).\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-8368946/v1/cc282ab8465ae19c4239f1a6.png"},{"id":98989082,"identity":"27f4d652-e15c-4fc5-880e-8c85866773e7","added_by":"auto","created_at":"2025-12-25 09:42:18","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1560890,"visible":true,"origin":"","legend":"\u003cp\u003eSAXS analysis of SDSD Psr and LytR LCP domains. (A) Psr envelope (light grey surface) overlaid with its AF2 model (cartoon) and fit of the corresponding model to the SAXS data. (B) LytR envelope (black surface) overlaid with its crystal structure (cartoon) and fit of the corresponding model to the SAXS data.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-8368946/v1/1418685e049fd61e67d9d61a.png"},{"id":98989063,"identity":"591a2bcd-d625-4bbc-893a-fc2a4cbea250","added_by":"auto","created_at":"2025-12-25 09:42:17","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":181810,"visible":true,"origin":"","legend":"\u003cp\u003eActivity of the Psr protein. (A) Malachite-green assay showing temperature-dependent conversion of ADP → AMP, detected via Pᵢ release. (B) ¹H NMR monitoring of AMP formation (dark-grey squares) and ADP consumption (light-grey dots) based on H8 chemical-shift intensities.\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-8368946/v1/82b311a068ee86c8f60172dc.png"},{"id":104297645,"identity":"de4b0ca0-20d1-43cc-ada4-91e213444608","added_by":"auto","created_at":"2026-03-10 08:12:50","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":8520529,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8368946/v1/9c0916f4-3c3a-4938-afa9-9fecdf3eeaaf.pdf"},{"id":99312266,"identity":"2de462f7-01fa-4e36-9dca-6c8d862b1685","added_by":"auto","created_at":"2025-12-31 16:18:32","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":11357896,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-8368946/v1/a2982d8d89636c6ee31445be.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Structural and Functional Insights into a Previously Uncharacterized Psr LCP Protein from Streptococcus dysgalactiae subsp. dysgalactiae","fulltext":[{"header":"Key points","content":"\u003cp\u003e\u0026bull; An unannotated SDSD protein is identified as Psr LCP by phylogenetics.\u003c/p\u003e\u003cp\u003e\u0026bull; Structural analyses reveal a conserved fold with Psr-specific features.\u003c/p\u003e\u003cp\u003e\u0026bull; A new \u0026sup1;H NMR assay enables real-time, label-free LCP kinetics.\u003c/p\u003e"},{"header":"Introduction","content":"\u003cp\u003eThe rise of antibiotic-resistant bacteria is one of the most pressing global health threats of our time (Frieri et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Miethke et al. 2021; Salam et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Sengupta et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Ventola \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The emergence of multidrug-resistant strains such as methicillin-resistant \u003cem\u003eStaphylococcus aureus\u003c/em\u003e (MRSA), vancomycin-resistant \u003cem\u003eEnterococci\u003c/em\u003e (VRE), and carbapenem-resistant \u003cem\u003eEnterobacteriaceae\u003c/em\u003e (CRE) has rendered many antibiotics ineffective (Frieri et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), underscoring the need to identify new therapeutic targets within essential bacterial pathways.\u003c/p\u003e \u003cp\u003eIn Gram-positive bacteria, cell-wall integrity and remodeling rely on the coordinated action of enzymes that synthesize, modify, and anchor glycopolymers to peptidoglycan (Do et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Rausch et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Among these, the LytR-CpsA-Psr (LCP) family, present in virtually all Gram-positive species, catalyzes the final transfer of wall teichoic acids (WTAs) and other cell-wall glycopolymers (CWGPs) onto the peptidoglycan backbone, a reaction essential for cell-wall assembly, division, and biofilm formation (Gale et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Harrison et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; H\u0026uuml;bscher et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Kawai et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Stefanović et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Because this process occurs on the outer surface of the cell membrane, LCP enzymes are accessible to inhibitors and have emerged as promising antimicrobial targets (Kawai et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Stefanović et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Yet, despite their physiological importance and widespread conservation, detailed structural and functional information for these proteins remains limited.\u003c/p\u003e \u003cp\u003eDuring biosynthesis, CWGPs are linked to an undecaprenyl carrier via a pyrophosphate bond (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). LCP proteins act as pyrophosphatases, cleaving this bond and transferring the CWGP from the lipid carrier to the peptidoglycan, forming a new phosphodiester linkage (Gale et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Harrison et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; H\u0026uuml;bscher et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Kawai et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Stefanović et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eDespite their variable topology and occasional accessory domains, and even with low sequence identity among homologs (including those within the same species) LCP proteins share a highly conserved overall fold. This includes a hydrophobic pocket that accommodates the lipid carrier and coordinates a divalent cation, typically Mg\u0026sup2;⁺, together with a pyrophosphate-containing substrate (H\u0026uuml;bscher et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Kawai et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Stefanović et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Although the pocket itself is universal, its residue composition tend to be conserved only within a given subfamily of proteins. Several subfamilies (LytR/BrpA, CpsA, Psr/LcpA, LcpB/TagT, LcpC/TagU and LcpD/TagV) are commonly found across Gram-positive species, often coexisting within the same genome (Kawai et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Schaefer et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Zilla et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2015b\u003c/span\u003e). Structural and biochemical data are available for members of the different subfamilies (Kawai et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Schaefer et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Siegel et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2019\u003c/span\u003e); however, only the LcpA/Psr protein from \u003cem\u003eS. aureus\u003c/em\u003e has been structurally characterized (Li et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), while the corresponding proteins from streptococcal species remain poorly characterized.\u003c/p\u003e \u003cp\u003eCrystal structures of LCP proteins with bound analogues of the lipidic carrier containing phosphate or diphosphate moieties reveal the charged portion of the substrate positioned near the pocket entrance, where catalysis occurs (Eberhardt et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Kawai et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Li et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Schaefer et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Four conserved arginine residues stabilize the negatively charged diphosphate group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), and their mutation (e.g., R118A, R219A, R227A in \u003cem\u003eBacillus subtilis\u003c/em\u003e TagT) abolishes activity (Schaefer et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Two conserved aspartates coordinate the Mg\u0026sup2;⁺ cofactor, which adopts an octahedral geometry completed by two water molecules and two substrate oxygens (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) (Kawai et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Schaefer et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The metal ion is essential for catalysis, as activity decreases sharply in the presence of chelators such as EDTA or upon mutation of either aspartate (Gale et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Kawai et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eStreptococcus dysgalactiae subsp. dysgalactiae\u003c/em\u003e (SDSD) is an opportunistic pathogen associated with bovine mastitis, fish streptococcosis, and, increasingly, human infections. Its genome encodes multiple LCP homologs, suggesting functional diversification related to cell-envelope remodeling and virulence. Because these enzymes operate extracellularly and catalyze an essential step in cell-wall assembly, they are considered promising antibiotic targets (H\u0026uuml;bscher et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Sewell and Brown \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). However, inhibition is complicated by the frequent presence of several partially redundant LCP genes within a single organism (Over et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). In many bacteria, single knockouts have only mild effects on growth or virulence, whereas multiple deletions lead to severe envelope defects or lethality (Dengler et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Over et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Understanding the specific structural and functional traits that distinguish individual LCP proteins is therefore critical for designing selective or synergistic inhibitors.\u003c/p\u003e \u003cp\u003eHere, we report the structural and functional characterization of two LCP proteins from \u003cem\u003eS. dysgalactiae subsp. dysgalactiae\u003c/em\u003e: LytR (previously described in (Paquete-Ferreira et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2024\u003c/span\u003e)) and a newly characterized Psr-type enzyme. Using phylogenetic analysis, AlphaFold2 modeling, and small-angle X-ray scattering (SAXS), we show that this enzyme adopts the canonical LCP fold but exhibits distinct structural features relative to LytR. Sequence analysis highlights residues potentially associated with Psr-specific substrate recognition. Enzymatic activity was confirmed by colorimetric and nuclear magnetic resonance (NMR) assays, demonstrating ADP-to-AMP conversion and metal dependence. Importantly, the continuous, label-free, \u0026sup1;H NMR approach introduced here provides a complementary tool for assessing LCP activity in real time and sets the stage for future kinetic and inhibitor studies.\u003c/p\u003e"},{"header":"Methods","content":"\u003ch2\u003eGenetic\u0026nbsp;construction\u0026nbsp;and site-directed mutagenesis\u003c/h2\u003e\n\u003cp\u003eThe construct for the LCP domain of the Psr protein from \u003cem\u003eStreptococcus dysgalactiae\u003c/em\u003e subsp. \u003cem\u003edysgalactiae\u003c/em\u003e (SDSD) was produced by amplifying the gene fragment encoding residues 213\u0026ndash;480, corresponding to the LCP domain, from genomic DNA (VSD9 isolate). The amplified fragment was cloned into the pNIC28-Bsa4 vector, which encodes an N-terminal hexahistidine tag followed by a TEV protease cleavage site, using the ligase-independent cloning (LIC) system. The primers used are listed in Table S1 (Supplementary Material).\u003c/p\u003e\n\u003cp\u003eThe construct excludes the membrane-anchoring helix located between the two domains to ensure soluble expression of the catalytic LCP region. The production of the LytR LCP domain construct was performed as previously described (Paquete-Ferreira et al. 2024).\u003c/p\u003e\n\u003ch2\u003eProtein expression and purification\u003c/h2\u003e\n\u003cp\u003eFor the production of the LCP domain of Psr, the construct was transformed into \u003cem\u003eE. coli\u003c/em\u003e BL21 (DE3) cells. Cultures were grown in LB medium supplemented with kanamycin (50 \u0026micro;g/mL) at 37 \u0026deg;C and 180 rpm. When the optical density (OD₆₀₀) reached 0.5\u0026ndash;0.8, protein expression was induced with 1 mM IPTG, and incubation continued overnight at 16 \u0026deg;C and 180 rpm.\u003c/p\u003e\n\u003cp\u003eCells were harvested by centrifugation (8,000 rpm, 10 min, 4 \u0026deg;C), resuspended in lysis buffer (10 mM K₂HPO₄ pH 6.0, 500 mM NaCl, 5 mM MgCl₂, and 10 mM imidazole), and disrupted by sonication (10 s on, 20 s off, 50 % amplitude) for 10 min. The lysate was clarified by centrifugation (11,000 \u0026times; g, 30 min, 6 \u0026deg;C), and the supernatant was loaded onto a 5 mL Ni\u0026sup2;⁺ HisTrap\u0026trade; HP column (Cytiva) for immobilized metal-affinity chromatography (IMAC). The protein was eluted using a linear imidazole gradient (30\u0026ndash;500 mM).\u003c/p\u003e\n\u003cp\u003eFractions containing the Psr LCP domain were dialyzed overnight at 4 \u0026deg;C against 1 L of 10 mM sodium citrate pH 6.0, 500 mM NaCl, and 5 mM MgCl₂ with gentle stirring. The dialyzed protein was concentrated and further purified by size-exclusion chromatography (SEC) using a Superdex 200 Increase 10/300 GL column (Cytiva). Fractions containing the monomeric protein were pooled, concentrated to 10 mg/mL, and stored at \u0026minus;20 \u0026deg;C.\u003c/p\u003e\n\u003cp\u003eThe LytR LCP domain was expressed and purified following the protocol previously described (Paquete-Ferreira et al. 2024).\u003c/p\u003e\n\u003ch2\u003eSmall-angle X-ray scattering (SAXS)\u003c/h2\u003e\n\u003cp\u003eSamples of the LytR and Psr LCP domains were prepared at concentrations of 7.7 mg/mL and 4.2 mg/mL, respectively, in 50 mM HEPES pH 8.0, 150 mM NaCl, and 5 mM MgCl₂, and subsequently subjected to five serial dilutions.\u003c/p\u003e\n\u003cp\u003eSAXS experiments were conducted at the European Synchrotron Radiation Facility (ESRF, Grenoble, France) on beamline BM29. Data inspection, reduction, and preliminary analysis were performed using PRIMUS\u0026nbsp;(Konarev et al. 2003), allowing determination of the radius of gyration (R\u003csub\u003eg\u003c/sub\u003e) and forward scattering intensity (I₀) from Guinier plots and pair-distance distribution functions. A summary of data collection and analysis parameters is provided in Table 1.\u003c/p\u003e\n\u003cp\u003eTheoretical scattering curves from the crystallographic and modeled structures were calculated and compared to the experimental profiles using Crysol and SREFLEX (Svergun et al. 1995). \u003cem\u003eAb initio\u003c/em\u003e shape reconstructions were generated with DAMMIF, and the resulting models were averaged and superimposed using DAMAVER (Franke and Svergun 2009; Panjkovich and Svergun 2016).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1\u003c/strong\u003e: SAXS samples and analysis\u003c/p\u003e\n\u003cp\u003ea. Sample details\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"100%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 36px;\"\u003e\n \u003cp\u003eOrganism\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 63px;\"\u003e\n \u003cp\u003e\u003cem\u003eStreptococcus dysgalactiae subsp. dysgalactiae\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 36px;\"\u003e\n \u003cp\u003eSource\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 63px;\"\u003e\n \u003cp\u003e\u003cem\u003eE. coli\u003c/em\u003e (BL21) recombinant expression\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 36px;\"\u003e\n \u003cp\u003eScattering particle composition\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 30px;\"\u003e\n \u003cp\u003eLytR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 32px;\"\u003e\n \u003cp\u003ePsr\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 36px;\"\u003e\n \u003cp\u003eProtein\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 30px;\"\u003e\n \u003cp\u003eLytR LCP domain\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 32px;\"\u003e\n \u003cp\u003ePsr LCP domain\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 36px;\"\u003e\n \u003cp\u003eLigand\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 63px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 36px;\"\u003e\n \u003cp\u003eStoichiometry of components\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 63px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 36px;\"\u003e\n \u003cp\u003eSolvent composition\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 63px;\"\u003e\n \u003cp\u003e50 mM HEPES pH 8.0, 150 mM NaCl and 5 mM MgCl\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 36px;\"\u003e\n \u003cp\u003eSample concentration (mg/ mL)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 30px;\"\u003e\n \u003cp\u003e7.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 32px;\"\u003e\n \u003cp\u003e4.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eb. SAS data collection\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"100%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 54px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 45px;\"\u003e\n \u003cp\u003eESRF BM29\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 54px;\"\u003e\n \u003cp\u003eData-acquisition/reduction software\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 45px;\"\u003e\n \u003cp\u003e\u003cem\u003eBSXCuBE\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 54px;\"\u003e\n \u003cp\u003eSource/instrument description\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 45px;\"\u003e\n \u003cp\u003e2 Pole Wiggler, Pilatus3 2M in-vacuo\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 54px;\"\u003e\n \u003cp\u003eMeasured q-range (q\u003csub\u003emin\u0026nbsp;\u003c/sub\u003e- q\u003csub\u003emax\u003c/sub\u003e) (\u0026Aring;\u003csup\u003e-1\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 45px;\"\u003e\n \u003cp\u003e0.0042 \u0026ndash; 0.522\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 54px;\"\u003e\n \u003cp\u003eExposure time (s), No. of exposures\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 45px;\"\u003e\n \u003cp\u003e10 frames\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ec. SAS-derived structural parameters\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"100%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 51px;\"\u003e\n \u003cp\u003eMethod(s)/software\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 48px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 51px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 23px;\"\u003e\n \u003cp\u003eLytR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 24px;\"\u003e\n \u003cp\u003ePsr\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 51px;\"\u003e\n \u003cp\u003eGuinier analysis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 23px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 24px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 51px;\"\u003e\n \u003cp\u003eI(0) \u0026plusmn;\u0026nbsp;𝜎\u0026nbsp;(cm\u003csup\u003e-1\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 23px;\"\u003e\n \u003cp\u003e14.86 \u0026plusmn; 0.079\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 24px;\"\u003e\n \u003cp\u003e9.58 \u0026plusmn; 0.083\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 51px;\"\u003e\n \u003cp\u003eRg \u0026plusmn;\u0026nbsp;𝜎(nm)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 23px;\"\u003e\n \u003cp\u003e2.50 \u0026plusmn; 0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 24px;\"\u003e\n \u003cp\u003e2.48 \u0026plusmn; 0.03\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 51px;\"\u003e\n \u003cp\u003eqRg range (datapoint range)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 23px;\"\u003e\n \u003cp\u003e0.41-1.30 (24-93)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 24px;\"\u003e\n \u003cp\u003e0.41-1.29 (25-93)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 51px;\"\u003e\n \u003cp\u003eLinear fit assessment (AUTORG fidelity)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 23px;\"\u003e\n \u003cp\u003e0.66\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 24px;\"\u003e\n \u003cp\u003e0.45\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 51px;\"\u003e\n \u003cp\u003ePDDF/P(r) analysis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 23px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 24px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 51px;\"\u003e\n \u003cp\u003eI(0) \u0026plusmn;\u0026nbsp;𝜎\u0026nbsp;(cm\u003csup\u003e-1\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 23px;\"\u003e\n \u003cp\u003e15.01 \u0026plusmn; 0.063\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 24px;\"\u003e\n \u003cp\u003e9.58 \u0026plusmn; 0.077\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 51px;\"\u003e\n \u003cp\u003eRg \u0026plusmn;\u0026nbsp;𝜎(nm)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 23px;\"\u003e\n \u003cp\u003e2.62 \u0026plusmn; 0.011\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 24px;\"\u003e\n \u003cp\u003e2.53 \u0026plusmn; 0.028\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 51px;\"\u003e\n \u003cp\u003edmax (nm)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 23px;\"\u003e\n \u003cp\u003e8.87\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 24px;\"\u003e\n \u003cp\u003e9.28\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 51px;\"\u003e\n \u003cp\u003eq-range (nm\u003csup\u003e-1\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 23px;\"\u003e\n \u003cp\u003e0.16-3.20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 24px;\"\u003e\n \u003cp\u003e0.16-3.25\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 51px;\"\u003e\n \u003cp\u003ep(r) reciprocal-space fit\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 23px;\"\u003e\n \u003cp\u003e0.83\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 24px;\"\u003e\n \u003cp\u003e0.78\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eMalachite Green Assay\u003c/h2\u003e\n\u003cp\u003eThe malachite green assay was used to monitor the pyrophosphatase activity of the Psr LCP domain using ADP as substrate, following the procedure previously described (Paquete-Ferreira et al. 2024). To determine the optimal temperature for enzymatic activity, the wild-type Psr LCP domain (42 \u0026micro;M) was incubated with ADP (750 \u0026micro;M) in 50 mM HEPES pH 8.0, 150 mM NaCl, and 5 mM MgCl₂. Reactions were carried out overnight at 4, 20, 37, 42, and 50 \u0026deg;C.\u003c/p\u003e\n\u003ch2\u003eNuclear Magnetic Resonance (NMR) spectroscopy\u003c/h2\u003e\n\u003cp\u003eThe enzymatic activity of the Psr LCP domain was also monitored by \u0026sup1;H NMR spectroscopy using ADP as substrate. The reaction was followed by acquiring a series of 1,500 consecutive \u0026sup1;H spectra (32 s each; total acquisition time \u0026asymp;13.3 h), measuring the intensity of the peaks corresponding to the H8 proton of the purine moiety of both ADP and AMP.\u003c/p\u003e\n\u003cp\u003eThe reaction mixture contained 75 \u0026micro;M protein and 1.5 mM ADP in 10 mM sodium citrate pH 6.0, 500 mM NaCl, 5 mM MgCl₂, 10 % D₂O, and 50 \u0026micro;M 4,4-dimethyl-4-silapentanesulfonic acid (DSS) as an internal chemical-shift reference. After preparation, the sample was transferred to a 3 mm NMR tube and immediately placed in the spectrometer; acquisition started approximately 255 s later. A control sample containing ADP without protein was analyzed under identical conditions.\u003c/p\u003e\n\u003cp\u003eNMR spectra were recorded at 293 K on a 600 MHz Bruker Avance III spectrometer (proton Larmor frequency 600.13 MHz) equipped with a 5 mm TCI cryoprobe. Data were processed using TopSpin 4.2.0 (Bruker BioSpin). All \u0026sup1;H spectra were acquired with a spectral width of 9615.39 Hz centered at 2806.21 Hz, using eight transients, 32k data points, and a relaxation delay of 1.0 s. Solvent suppression was achieved by excitation sculpting with gradients (Hwang and Shaka 1995), applying a selective pulse (Squa100.1000) of 2 ms duration to irradiate the solvent signal.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eSDSD Psr clusters within the Psr/LcpA subfamily\u003c/h2\u003e \u003cp\u003eA common feature of the LCP protein family is the presence of multiple genes encoding this domain. In SDSD, there are two: the previously described LytR/BrpA (Paquete-Ferreira et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) and a second, uncharacterized protein analyzed here (Psr). The two share low overall sequence identity (12.9%), with the LCP domains showing 21.9% identity, a divergence similar to that observed among LCP paralogs in other species such as \u003cem\u003eBacillus subtilis\u003c/em\u003e, \u003cem\u003eStreptococcus pneumoniae\u003c/em\u003e, and \u003cem\u003eStaphylococcus aureus\u003c/em\u003e. The proteins also differ topologically: while LytR/BrpA includes a membrane-anchoring helix and two extracellular domains (the LCP core and a C-terminal accessory domain), Psr contains an intracellular N-terminal region, a transmembrane helix, and a single extracellular LCP domain.\u003c/p\u003e \u003cp\u003eTo examine the evolutionary relationship of SDSD Psr within the LytR-CpsA-Psr (LCP) family, its sequence was aligned with representative LCP genes from \u003cem\u003eB. subtilis\u003c/em\u003e, (proteins categorized as TagTUV), \u003cem\u003eS. aureus\u003c/em\u003e, \u003cem\u003eBacillus anthracis\u003c/em\u003e (proteins categorized as LcpA, LcpB, LcpC and LcpD), \u003cem\u003eS. pneumoniae\u003c/em\u003e, \u003cem\u003eS. agalactiae\u003c/em\u003e and \u003cem\u003eS. mutans\u003c/em\u003e (proteins categorized as LytR/BrpA, CpsA and Psr). Multiple sequence alignment (ClustaL Omega (Sievers and Higgins 2017)) indicated that SDSD Psr retains the conserved motifs characteristic of LCP enzymes, consistent with the presence of key catalytic residues.\u003c/p\u003e \u003cp\u003ePhylogenetic analysis performed using IQ-TREE web server with 1,000 ultrafast bootstraps (Trifinopoulos et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), placed SDSD Psr within the Psr/LcpA subfamily, clustering closely with other streptococcal homologs and clearly separating from LytR-type enzymes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). These results agree with those by L. Zilla and colleagues (Zilla et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2015a\u003c/span\u003e). Specifically, \u003cem\u003eB. subtilis\u003c/em\u003e TagT clusters with \u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eB. anthracis\u003c/em\u003e LcpB proteins, while \u003cem\u003eB. subtilis\u003c/em\u003e TagU is in the same branch as \u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eB. anthracis\u003c/em\u003e LcpC proteins. Additionally, \u003cem\u003eB. subtilis\u003c/em\u003e TagV clusters with \u003cem\u003eB. anthracis\u003c/em\u003e LcpD, and \u003cem\u003eS. aureus\u003c/em\u003e LcpA is found on a separate branch. By contrast, proteins characterized as LytR/BrpA and CpsA form branches distinct from the LcpB/C/D clades.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe uncharacterized SDSD protein, as well as all proteins categorized as Psr, are clustered within the same branch as \u003cem\u003eS. aureus\u003c/em\u003e LcpA. This observation aligns with existing NCBI annotations, where \u003cem\u003eS. aureus\u003c/em\u003e LcpA is described as MsrR - a designation similar to \u003cem\u003eS. mutans\u003c/em\u003e Psr. Together with the observed topology and the fact that \u003cem\u003eS. aureus\u003c/em\u003e LcpA can also be categorized as a Psr protein, this supports its classification as a Psr-type protein and suggests functional similarity to LcpA-like enzymes described in \u003cem\u003eStaphylococcus aureus\u003c/em\u003e and \u003cem\u003eBacillus subtilis\u003c/em\u003e. This classification is consistent with phylogenetic evidence and reduces potential confusion. Additionally, our analysis identified \u003cem\u003eS. agalactiae\u003c/em\u003e CAD46000 as a LytR/BrpA protein, contributing to a clearer understanding of LCP protein classification within these species.\u003c/p\u003e \u003cp\u003eAccording to this analysis, all \u003cem\u003eStreptococcus\u003c/em\u003e species contain a LytR protein. Notably, \u003cem\u003eS. agalactiae\u003c/em\u003e is the only \u003cem\u003eStreptococcus\u003c/em\u003e species that lacks a Psr protein from the group under study. Also, along with \u003cem\u003eS. pneumoniae\u003c/em\u003e, this is one of the only two species encoding CpsA proteins. Therefore, the data suggest that in the Streptococcus genus, LytR is likely essential, Psr may serve a comparable role, and CpsA appears to have a more specialized function, consistent with its known involvement in capsule synthesis and transfer.\u003c/p\u003e \u003cp\u003eCollectively, these findings indicate that Psr proteins diverged early from the rest of the LCP family, reflecting an ancient evolutionary split. Sequence similarities are stronger among functional subtypes than among species, implying that each LCP class has been shaped by similar selective pressures. This observation supports the hypothesis that horizontal gene transfer has contributed to the distribution and diversification of LCP enzymes.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePredicted structure shows a conserved LCP fold\u003c/h3\u003e\n\u003cp\u003eTo predict the 3D structure of the designed SDSD Psr construct and compare it with related proteins, we modeled the Psr LCP domains from \u003cem\u003eStreptococcus pyogenes\u003c/em\u003e, \u003cem\u003eS. mutans\u003c/em\u003e, and \u003cem\u003eS. pneumoniae\u003c/em\u003e identified in the phylogenetic analysis using AlphaFold 2 (AF2) (Jumper et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). All structures yielded high-confidence predictions for the LCP domain with a per-residue confidence (pLDDT)\u0026thinsp;\u0026gt;\u0026thinsp;90 for most of the core region (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The SDSD Psr model displays the canonical LCP architecture, consisting of a mixed α/β-fold in which a central β-sheet is surrounded by α-helices forming a compact catalytic core. The predicted topology includes a single transmembrane helix that connects to the LCP domain by a flexible region, consistent with other LCP enzymes (Stefanović et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSequence identities and RMSD values of the Psr LCP domains (including LytR) relative to the SDSD Psr LCP domain, as well as hydrophobic-pocket volumes calculated with CASTp (Tian et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eComparison of sequence identity, RMSD values and the pocket volume of SDSD Psr with the other identified Psr proteins and SDSD LytR\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eProtein\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSequence identity relative to SDSD Psr (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRMSD relative to SDSD Psr (aligned Cα)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePocket volume (\u0026Aring;\u003csup\u003e3\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSDSD Psr\u003c/p\u003e \u003cp\u003e(AF2 model)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e652\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSDSD LytR\u003c/p\u003e \u003cp\u003e(PDB ID: 8QTY)(Paquete-Ferreira et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2024\u003c/span\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e24.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.72 \u0026Aring; (150/150)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e411\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eS. pyogenes\u003c/em\u003e Psr\u003c/p\u003e \u003cp\u003e(AF2 model)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e75.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.39 \u0026Aring; (211/211)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e561\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eS. pneumoniae\u003c/em\u003e Psr\u003c/p\u003e \u003cp\u003e(AF2 model)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e54.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.55 \u0026Aring; (202/202)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e521\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eS. mutans\u003c/em\u003e Psr\u003c/p\u003e \u003cp\u003e(AF2 model)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e63.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.47 \u0026Aring; (210/210)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e609\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eS. aureus\u003c/em\u003e LcpA\u003c/p\u003e \u003cp\u003e(PDB ID: 6UEX)(Li et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2020\u003c/span\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e32.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.09 \u0026Aring; (174/174)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e549\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eAnalysis of these data shows that the streptococcal Psr LCP domains share higher sequence identity (54.2\u0026ndash;75.0%) with SDSD Psr than either SDSD LytR (24.2%) or \u003cem\u003eS. aureus\u003c/em\u003e LcpA (32.2%), a trend also reflected in their lower RMSD values. These results reinforce the phylogenetic grouping described above. Pocket-volume measurements do not reveal a consistent pattern, displaying variable values even among the streptococcal Psr LCP domains.\u003c/p\u003e \u003cp\u003eGiven the high similarity among Psr LCP domains, we performed a multiple-sequence alignment followed by structural comparison to evaluate whether the variability observed across the LCP family is reduced within this subgroup. The alignment included all Psr LCP domains and SDSD LytR, while the structural analysis used the SDSD Psr AF2 model, the SDSD LytR crystal structure (PDB ID: 8QTY (Paquete-Ferreira et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2024\u003c/span\u003e)), and the \u003cem\u003eS. aureus\u003c/em\u003e LcpA structure (PDB ID: 6UEX (Li et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2020\u003c/span\u003e)) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Although residue variability is distributed throughout the structure, we focused on differences at the peptidoglycan-binding site and within the hydrophobic pocket, where some hydrophobic residues, despite maintaining similar chemical properties, are not fully conserved.\u003c/p\u003e \u003cp\u003eWithin the peptidoglycan-binding site, residue R235 (SDSD Psr numbering) appears to be unique to Psr proteins, being replaced by an asparagine in SDSD LytR. In contrast, residues K278 and D392 are more broadly conserved between both LCP sub-types. Similarly, in the hydrophobic pocket, residues F317 and F320 are conserved among Psr proteins but replaced by methionine and leucine, respectively, in SDSD LytR, whereas L380 remains unchanged.\u003c/p\u003e \u003cp\u003eThe sequence alignment (Figure S2) also revealed a motif distinguishing LytR and Psr proteins: LytR enzymes contain RMR(X)₆DXGR, whereas Psr proteins exhibit RFR(X)₅DXGR. The substitution of methionine for phenylalanine and the one-residue shorter spacing likely influence substrate binding, suggesting distinct preferences for peptidoglycan, at different maturation stages, between the two LCP subtypes.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003ePsr displays the canonical LCP architecture in solution\u003c/h2\u003e \u003cp\u003eTo experimentally validate the AF2 model and compare it with LytR, we performed small-angle X-ray scattering (SAXS) analysis. Initial data reduction and preliminary analysis revealed concentration-dependent effects in the scattering profiles, reflected in variations of the radius of gyration (R\u003csub\u003eg\u003c/sub\u003e). Nevertheless, R\u003csub\u003eg\u003c/sub\u003e and forward scattering intensity (I₀) values could be reliably extracted for both proteins (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). To determine the pair-distance distribution functions p(r), selected curves were merged by combining low-angle data from the lowest concentration with high-angle data from the highest concentration. This approach yielded D\u003csub\u003emax\u003c/sub\u003e values. Figure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e shows the merged scattering curves for LytR and Psr (A) and their corresponding p(r) functions (B).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe comparison of the SAXS profiles indicates that the two proteins display broadly similar scattering behavior, with a χ\u0026sup2; value of 2.40. The main differences occur at higher angles, suggesting an overall comparable architecture with local structural variations. These may arise from the His-tags being positioned at opposite termini: C-terminal in LytR and N-terminal in Psr. The p(r) functions further suggest a slightly more elongated structure for Psr and a more flexible conformation for LytR.\u003c/p\u003e \u003cp\u003eFollowing the methodology previously described (Paquete-Ferreira et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), the experimental SAXS data were compared with theoretical scattering curves generated from the LytR crystal structure (PDB ID: 8QTY (Paquete-Ferreira et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2024\u003c/span\u003e); missing regions modelled with MODELLER) and the Psr AF2 model. The theoretical fits showed moderate agreement (χ\u0026sup2; = 2.82 for LytR and 2.36 for Psr). Refinement with SREFLEX improved the fits (χ\u0026sup2; = 2.44 and 2.08, respectively) and yielded RMSD values of 5.07 \u0026Aring; for LytR and 5.92 \u0026Aring; for Psr. In both cases, the global fold was preserved in solution, with the largest deviations occurring at the N-terminus of Psr and the C-terminus of LytR.\u003c/p\u003e \u003cp\u003e \u003cem\u003eAb-initio\u003c/em\u003e envelopes were generated for both proteins, yielding χ\u0026sup2; values of 0.98 for Psr and 1.13 for LytR, indicating excellent agreement between data and models. Figure\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e shows the averaged DAMFILT envelopes produced with DAMAVER, the corresponding best DAMMIF models, and their superpositions with the respective structures, calculated using CIFSUP.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eBoth Psr and LytR fit well within their SAXS envelopes, occupying most of the reconstructed volume. This correspondence indicates minimal conformational differences between the solution and modeled structures, confirming their structural stability in solution.\u003c/p\u003e \u003cp\u003eNevertheless, subtle differences distinguish the two proteins. The Psr envelope exhibits a shallow cleft that may correspond to a flexible or interaction region, whereas the LytR envelope appears more compact and uniform, suggesting a more rigid structure. Additionally, one loop near the cleft in the Psr model adopts a different conformation from that in LytR, potentially contributing to its greater flexibility or accessibility. Neither protein displays a strictly globular shape; both include an additional, smaller region that imparts asymmetry, though its position differs between the two. These observations are consistent with our previous results (Paquete-Ferreira et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), supporting the view that Psr and LytR share a similar overall conformation, with the main differences localized to mobile or flexible regions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eFunctional characterization of the Psr LCP domain\u003c/h2\u003e \u003cp\u003eTo assess the enzymatic activity of the SDSD Psr LCP domain, we monitored the conversion of ADP into AMP using both the malachite green assay and \u0026sup1;H NMR spectroscopy. In our previous work on the SDSD LytR domain (Paquete-Ferreira et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), ADP was introduced as a soluble substrate for evaluating LCP pyrophosphatase activity; here, we extend that concept by using \u0026sup1;H NMR to monitor the reaction directly in solution.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eTemperature dependence\u003c/strong\u003e \u003cp\u003eThe optimal temperature for Psr activity was determined by malachite green assay at five temperatures (4, 20, 37, 42, and 50\u0026deg;C). The reactions were incubated overnight, with samples taken at the start and end points (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). The enzyme displayed maximal activity at 37\u0026deg;C, achieving 77.2% conversion. Notably, Psr remained active up to 50\u0026deg;C, with conversions of 18.4% (4\u0026deg;C), 34.1% (20\u0026deg;C), 49.2% (42\u0026deg;C), and 49.0% (50\u0026deg;C). These results indicate good thermal stability across a range of temperatures and a clear preference for higher temperatures.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eNMR assay for real-time monitoring\u003c/strong\u003e \u003cp\u003eTo follow the reaction continuously, we implemented a label-free \u0026sup1;H NMR assay. Comparison of the 1D spectra of ADP and AMP (Figure S3) revealed distinct chemical shifts for the H8 proton of the purine moiety, enabling real-time tracking of substrate consumption and product formation (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB).\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNotably, the \u0026sup1;H NMR spectra already exhibited a small AMP signal at the start of the reaction, attributed to trace AMP present in the ADP reagent. This baseline signal was constant across all samples. During the enzymatic reaction, the AMP peak increased steadily, reflecting conversion of ADP to AMP. Control experiments confirmed that, in the absence of enzyme, the AMP signal did not change (Figure S3), demonstrating that ADP does not undergo spontaneous hydrolysis under the assay conditions. Although the optimal activity was observed at 37\u0026deg;C, NMR experiments were conducted at 20\u0026deg;C to prevent protein precipitation inside the NMR tube. Under these conditions, ~\u0026thinsp;25% conversion of ADP to AMP was achieved.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eKinetic behavior and inhibition\u003c/strong\u003e \u003cp\u003eContinuous monitoring by \u0026sup1;H NMR showed a linear evolution of ADP and AMP signals over time suggesting that, under the experimental conditions used, the reaction proceeds in an apparent zero-order regime. This steady-state behaviour is consistent with substrate saturation and demonstrates that the assay quantitatively captures catalytic progression.\u003c/p\u003e \u003c/p\u003e \u003cp\u003eTo validate the assay and assess inhibition, two negative controls were performed: (i) in the absence of enzyme and (ii) in the presence of enzyme with EDTA (Figure S3). Although EDTA did not fully inhibit activity, a clear reduction was observed, consistent with Mg\u0026sup2;⁺ chelation and the reported metal dependence of LCP enzymes (Kawai et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). We note that the use of a citrate buffer at pH 6.0 with 5 mM MgCl₂ likely reduced the effective chelating capacity of EDTA; however, this limitation does not affect the interpretation of the observed partial inhibition, which remains fully consistent with a Mg\u0026sup2;⁺-dependent catalytic mechanism.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis work combines phylogenetic, structural, and biochemical analyses to characterize a Psr-type LCP protein from \u003cem\u003eStreptococcus dysgalactiae\u003c/em\u003e subsp. \u003cem\u003edysgalactiae\u003c/em\u003e (SDSD). By integrating computational modeling, SAXS, and real-time NMR assays, we provide new insights into the structural conservation, catalytic properties, and metal dependence of this enzyme. The results highlight both the shared and distinctive features of Psr relative to other LCP subfamilies and establish a methodological basis for quantitative, label-free monitoring of LCP activity.\u003c/p\u003e \u003cp\u003eThe phylogenetic analysis of the LCP family provides valuable insights into the classification and functional diversity of these enzymes. Our results support the division of the family into six sub-types: LytR/BrpA, CpsA, Psr/LcpA, LcpB, LcpC, and LcpD. These sub-types form well-defined and independently supported branches in the phylogenetic tree, with additional members clustering consistently within each subtype. This organization offers a practical framework for classifying uncharacterized LCP proteins, complementing existing nomenclature and facilitating the identification of new sub-families that expand our understanding of LCP functional diversity.\u003c/p\u003e \u003cp\u003eWithin this framework, LytR, LcpB, LcpC, and LcpD proteins appear more closely related to each other, whereas CpsA and especially Psr show greater divergence from the rest of the family. The early divergence of the Psr subgroup suggests unique evolutionary pressures or functional adaptations, possibly related to differences in the glycopolymers transferred to the peptidoglycan. Further structural and biochemical studies are required to determine whether this divergence reflects substrate specificity or variations in catalytic function.\u003c/p\u003e \u003cp\u003eThe SAXS data indicate that the LCP domains of LytR and Psr share an overall similar structure, with no evidence of significant disorder in either protein. This indicates that identical buffer conditions did not affect their global architecture, despite the different theoretical pI values of LytR (6.25) and Psr (5.02). The slightly higher D\u003csub\u003emax\u003c/sub\u003e observed for Psr points to a more elongated structure, possibly due to increased flexibility of the His-tag at its N-terminus compared with the C-terminal tag of LytR.\u003c/p\u003e \u003cp\u003eThe use of \u0026sup1;H NMR to monitor nucleotide conversions, particularly the hydrolysis of ATP to ADP, is well established (Lian et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Similar to earlier ATPase studies, the reaction was monitored through the distinct chemical shifts of the H8 proton of the purine ring (Guo et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). A key advantage of \u0026sup1;H NMR over the malachite-green assay is its ability to collect multiple time points from the same sample throughout the reaction, eliminating the need for larger reaction volumes. Moreover, \u0026sup1;H NMR allows simultaneous observation of substrate depletion and product formation, providing a more comprehensive view of reaction progress. Both techniques yielded comparable conversions, with differences explained by reaction time and buffer conditions. At 20\u0026deg;C, the malachite-green assay yielded 34.1 % conversion, while \u0026sup1;H NMR showed 5.4 %. This difference can largely be ttributed to the assay duration (\u0026asymp;\u0026thinsp;18 h versus 13 h). Assuming the apparent zero-order kinetics observed, a 5-h extension could account for an additional\u0026thinsp;~\u0026thinsp;9 % conversion, consistent with the iscrepancy. Interestingly, when applying these kinetics to estimate reaction velocities, \u0026sup1;H NMR yields 0.49 \u0026micro;M min⁻\u0026sup1; and the malachite-green assay 0.24 \u0026micro;M min⁻\u0026sup1;. The difference likely reflects the lower substrate excess (18-fold versus 20-fold) and the distinct pH conditions used in the two assays (pH 6.0 and 8.0, respectively).\u003c/p\u003e \u003cp\u003eIn a study by Schaefer and colleagues (Schaefer et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), the enzymatic activity of \u003cem\u003eB. subtilis\u003c/em\u003e TagT was also analyzed assuming a zero-order kinetic regime, in line with our findings. Nevertheless, the reaction investigated by Schaefer and colleagues involved glycopolymer transfer, whereas the present work focuses on ADP hydrolysis. Additional studies at lower substrate concentrations will be required to confirm whether the apparent zero-order behaviour reflects enzyme saturation in the ADP to AMP reaction or is a general kinetic feature of the LCP family. For the transfer reaction itself, continuous assays similar to the present one will help clarify whether it also proceeds at a constant rate.\u003c/p\u003e \u003cp\u003eThe EDTA inhibition assay showed only partial activity reduction. This outcome is consistent with previous reports (Kawai et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2011\u003c/span\u003e) but may also result from the buffer composition used here (10 mM sodium citrate pH 6.0, 500 mM NaCl, 1 mM EDTA). The relatively acidic pH likely decrease the effective chelating capacity of EDTA, allowing residual enzyme activity. Importantly, this limitation does not alter the interpretation of the results, which remain fully consistent with a Mg\u0026sup2;⁺-dependent catalytic mechanism.\u003c/p\u003e \u003cp\u003eOverall, the combined data demonstrate that the SDSD Psr protein adopts a conserved LCP fold, is catalytically active \u003cem\u003ein vitro\u003c/em\u003e, and exhibits robust thermal stability and metal dependence. The continuous \u0026sup1;H NMR assay introduced here provides a complementary, label-free approach for quantifying LCP activity in real time, paving the way for future kinetic and inhibition studies within this enzyme family.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to thank the staff of beamline BM29 (ESRF) for their assistance during the SAXS data collection.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJo\u0026atilde;o Paquete-Ferreira: Investigation; methodology; analysis; writing \u0026ndash; original draft; writing, review and editing\u003c/p\u003e\n\u003cp\u003eLeonor Torres: Investigation; methodology\u003c/p\u003e\n\u003cp\u003eJoana Bryton: Investigation; methodology\u003c/p\u003e\n\u003cp\u003eMarino F. A. Santos: Methodology; analysis; writing, review and editing\u003c/p\u003e\n\u003cp\u003eM\u0026aacute;rcia A. S. Correia: Methodology; analysis; writing, review and editing\u003c/p\u003e\n\u003cp\u003eAlexandra R. Fernandes: Supervision; resources; project administration; writing, review and editing\u003c/p\u003e\n\u003cp\u003eMaria Jo\u0026atilde;o Rom\u0026atilde;o: Supervision; resources; project administration; writing, review and editing\u003c/p\u003e\n\u003cp\u003eAldino Viegas: Investigation; methodology; analysis; writing, review and editing\u003c/p\u003e\n\u003cp\u003eTeresa Santos-Silva: Conceptualization; supervision; resources; project administration; analysis; writing, review and editing\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work is financed by national funds from FCT - Funda\u0026ccedil;\u0026atilde;o para a Ci\u0026ecirc;ncia e a Tecnologia, I.P., in the scope of the project UIDP/04378/2020 (DOI: 10.54499/UIDP/04378/2020) and UIDB/04378/2020 (DOI: 10.54499/UIDB/04378/2020) of the Research Unit on Applied Molecular Biosciences - UCIBIO and the project LA/P/0140/2020 (DOI: 10.54499/LA/P/0140/2020) of the Associate Laboratory Institute for Health and Bioeconomy - i4HB., through grant 2020.08580.BD (to J.P.F.), and contracts 2020.00043.CEECIND and 2023.11076TENURE.002 (to A.V.).\u003c/p\u003e\n\u003cp\u003eThe NMR spectrometer at NOVA-FCT is part of the National NMR Network (PTNMR) and is supported by national funds from FCT-MCTES through the scope of projects UIDB/04378/ 2020 of the Research Unit on Applied Molecular Biosciences (UCIBIO) and the project UID/50006/2023 of the Associate Laboratory for Green Chemistry (LAQV-REQUIMTE).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to thank the staff of beamline BM29 (ESRF) for their assistance during the SAXS data collection.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis study did not involve human participants or animals; therefore, ethical approval was not required.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAll authors certify that they have no affiliations with or involvement in any organization or entity with any financial interest or non-financial interest in the subject matter or materials discussed in this manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eDengler V, Meier PS, Heusser R, Kupferschmied P, Fazekas J, Friebe S et al (2012) Deletion of hypothetical wall teichoic acid ligases in Staphylococcus aureus activates the cell wall stress response. 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J Bacteriol 197(23):3731\u0026ndash;3741\u003c/span\u003e\u003c/li\u003e\u003c/ol\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":"Psr LCP protein, Streptococcus dysgalactiae, Antibiotic resistance, Small-angle X-ray scattering, Nuclear Magnetic Resonance, Protein structure and function","lastPublishedDoi":"10.21203/rs.3.rs-8368946/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8368946/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThere is a pressing need to develop new antibiotics targeting alternative biological pathways to effectively tackle the problem of antibiotic resistance. LytR-CpsA-Psr (LCP) proteins catalyze the attachment of cell-wall glycopolymers to peptidoglycan, a crucial step in Gram-positive bacterial envelope biogenesis and a promising target for antimicrobial development.\u003c/p\u003e \u003cp\u003eHere we report the structural and functional characterization of a Psr-type LCP protein from \u003cem\u003eStreptococcus dysgalactiae\u003c/em\u003e subsp. \u003cem\u003edysgalactiae\u003c/em\u003e (SDSD). Phylogenetic analysis places this enzyme within the Psr/LcpA clade, showing higher conservation among streptococcal homologs than with LytR-type enzymes. The AlphaFold2 model and small-angle X-ray scattering (SAXS) data reveal a conserved overall fold with subtle differences from LytR, including an elongated shape and flexible termini. Sequence analysis highlights distinctive Psr-specific residues (R235, F317, F320) that may influence substrate recognition.\u003c/p\u003e \u003cp\u003eEnzymatic activity was confirmed using both colorimetric and nuclear magnetic resonance (NMR) assays, demonstrating conversion of ADP to AMP with maximal activity near 37\u0026deg;C and an apparent zero-order kinetic regime under saturating substrate conditions. The \u0026sup1;H NMR assay, implemented here for the first time, provides a continuous, label-free approach to monitor LCP activity in real time. Together, these findings establish the structural conservation and functional distinctiveness of the SDSD Psr LCP domain, introduce a complementary NMR-based strategy suitable for kinetic and inhibitor studies within the LCP family and lay the groundwork for a deeper understanding of LCP-mediated processes, supporting their potential as antibiotic targets.\u003c/p\u003e","manuscriptTitle":"Structural and Functional Insights into a Previously Uncharacterized Psr LCP Protein from Streptococcus dysgalactiae subsp. dysgalactiae","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-25 09:42:12","doi":"10.21203/rs.3.rs-8368946/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":"6509cdde-7f34-4e15-856e-02a49483ea4c","owner":[],"postedDate":"December 25th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-03-10T08:11:57+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-25 09:42:12","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8368946","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8368946","identity":"rs-8368946","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}