Sequence Analysis of P4-ATPases Reveals the Structural Determinants for the Stable Monomeric P4B-ATPase Phospholipid Transporters

Sequence Analysis of P4-ATPases Reveals the Structural Determinants for the Stable Monomeric P4B-ATPase Phospholipid Transporters | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (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];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Sequence Analysis of P4-ATPases Reveals the Structural Determinants for the Stable Monomeric P4B-ATPase Phospholipid Transporters Kadambari Vijay Sai , Sai Anvithaa Sundar Rajan , View ORCID Profile Jyh-Yeuan Lee doi: https://doi.org/10.1101/2025.07.01.662597 Kadambari Vijay Sai 1 Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa , Ottawa, Ontario, Canada 2 Centre for Biotechnology, Anna University , Chennai, India Find this author on Google Scholar Find this author on PubMed Search for this author on this site Sai Anvithaa Sundar Rajan 1 Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa , Ottawa, Ontario, Canada 2 Centre for Biotechnology, Anna University , Chennai, India 3 Startycon Business Solutions Pvt limited Find this author on Google Scholar Find this author on PubMed Search for this author on this site Jyh-Yeuan Lee 1 Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa , Ottawa, Ontario, Canada Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Jyh-Yeuan Lee For correspondence: Jyh-Yeuan.Lee{at}uOttawa.ca Abstract Full Text Info/History Metrics Preview PDF ABSTRACT The P4-ATPase family of phospholipid flippases plays a critical role in the maintenance of membrane asymmetry and consequently, various roles in cellular protein traffic and eukaryotic homeostasis. Currently, several structures of these (usually heterodimeric) phospholipid flippases have been resolved, along with extensive biochemical characterization of the substrate transport properties. However, an essential subfamily of monomeric phospholipid flippases, the P4B-ATPases, remains to be characterized in depth. While these P4B-ATPases appear to have similar lipid transport properties to their heterodimeric counterparts, the P4A-ATPases, the basis of their existence as monomers is currently unknown. In addition, the unique features of this group have yet to be comprehensively analyzed since the resolution of one P4B-ATPase member. In this study, we investigated the divergence of P4B-ATPases from other P-type ATPases using a structure-based sequence analysis approach. This led to identification of features unique to P4B-ATPases, as well as previously unidentified conserved properties of P4-ATPases. The results of this study provide a basis for further studies on P4-ATPases to characterize conserved properties of this group that supersedes substrate specificities. MANUSCRIPT INFORMATION - Number of manuscript pages: 20 (double-spaced). - Number of Figures: 5. - Number of Tables: 3. - Supplementary materials include 2 supplementary tables. 1. INTRODUCTION P-type ATPases constitute a large and essential family of membrane transporters in eukaryotic and prokaryotic organisms. This family of proteins hydrolyze ATP to generate a phosphorylated intermediate that can perform active substrate transport across the membrane. They transport a variety of substrates ranging from ions, phospholipids to even transmembrane helices of membrane proteins. Depending on the substrate transported, they are classified into subfamilies from P1- to P5-ATPases, with further classification into subclasses due to substrate specificity and sequence divergence. Specifically in humans, the P-type ATPases are divided into P1B (copper), P2A (calcium), P2B (calmodulin), P2C (sodium-potassium), P4A (phospholipid), P4B (phospholipid), P5A (transmembrane helices) and P5B (polyamine) (For detailed reviews, ( Pedersen et al. 2012a ; Palmgren 2023 ; Stock et al. 2023 ). These P-type ATPases possess a similar overall architecture – a transmembrane domain with 10-12 transmembrane helices and three cytosolic domains – a nucleotide binding (N) domain, a phosphorylation (P) domain and an actuator (A) domain. These proteins undergo the Post-Albers reaction cycle to transport substances across the membrane bilayer in an ATP-dependant manner. This cycle involves phosphorylation and dephosphorylation of the protein, which is coupled to transport of substrates. The phosphorylated and dephosphorylated intermediates are open to opposite sides of the membrane (E1 – outward facing and E2 – inward facing) allowing substrate binding, occlusion and movement due to the progression of the reaction cycle. The P1, P2 and P3-ATPases transport substances in both the phosphorylation and dephosphorylation phases of the reaction cycle while P4 and P5-ATPases, which are specific to eukaryotes, transport their substrates only in the dephosphorylation phase of the cycle. (For further details of the Post-Albers reaction cycle in P4 and P-type ATPases refer recent reviews ( Norris et al. 2024 ; Duan and Li 2024 ; Sai and Lee 2024 ; Stock et al. 2023 ; Palmgren 2023 )) Several structural studies of P-type ATPases have been conducted in the past few decades. Different ligands have been used to generate specific intermediates of the Post-Albers cycle. ATP-bound conformations have been generated using ATP and ATP analogs such as AMPPCP, AMPPNP and ATPgS. Various phosphorylated intermediates have also been resolved. This includes E1P-states resolved using ADP and aluminium fluoride, E2P-states resolved using beryllium fluoride and the post-hydrolytic, substrate-occluded E2-Pi states resolved using MgF4 and VO 3 4- . Of note, there has been a huge influx of P4A-ATPase structures in the past few years, leading to a deep understanding of the structural and biochemical properties of their function. Meanwhile, the P4B-ATPases lack the same depth of biochemical and structural characterization. P4B-ATPases, which exhibit varying degrees of essentiality in eukaryotes, are predicted to have evolved earlier, where as P4A and P4C-ATPases are believed to have developed the requirement for a beta-subunit through evolution. This group includes flippases such as Neo1 of Saccharomyces cerevisiae , TAT-5 of Caenorhabditis elegans and ATP9A and ATP9B of Homo sapiens. An interesting aspect is that several residues of P4B-ATPases align structurally with other groups but not in multiple sequence alignment. The P4B-ATPases exhibit a high degree of sequence homology to one another, but a low degree to other P4-ATPases. Despite their monomeric nature, P4B-ATPases are believed to have a similar substrate translocation pathway as P4A-ATPases. The entry site residues are analogous to those present in other P4-ATPases and the exit site compensates for beta-subunit residues with those from the alpha-subunit. The stability of P4B and ability to maintain a conserved translocation mechanism with a reduced length and absence of a beta-subunit remain elusive. It has been suggested that they evolved additional extracellular loops to bind the beta-subunit. However, the structural features which allow them to exist as a monomer remain to be conclusively determined. To investigate the sequence determinant and the structural basis for P4B-ATPase monomeric stability, we performed a comprehensive sequence analysis of P4B-ATPases and P4-ATPases in comparison with other P-type ATPase subfamilies, aiming to determine whether they inherited their monomeric nature from the other monomeric P-type ATPases or whether they possess unique characteristics that allow them to exist as a monomer. This work identified several features characteristic of P4-ATPases and those that distinguish P4A- and P4B-ATPases. This study will provide important insights into the structural evolution of P4-ATPases and lay the foundation for future mechanistic investigations of these essential membrane transporters. 2. RESULTS 2.1. Conserved residues in the P4-ATPase transporters In this study, all the human P-type ATPases, in addition to the P4-ATPases from Saccharomyces cerevisiae were used. The plant P4-ATPases were also utilized in this study to ensure representation of the P4C subclass, often absent in fungi and mammals. Due to the high level of sequence identity among the plant P4-ATPases ( Fig. 1A ) and the relatively fewer studies conducted on their biochemical properties, the alignments of plant proteins were primarily used to verify conservation of residues in the P4-ATPase family. Additionally, they were also used to understand sequence divergence between the heterodimeric and monomeric P4-ATPases. Download figure Open in new tab Figure 1. Sequence identity and structural similarity in the P-type ATPase family. (A) Heatmap indicating the level of sequence identity between the various members of the P-type family in humans along with P4-ATPases from Saccharomyces cerevisiae and Arabidopsis thaliana . (B) Phylogenetic tree showing the classification of P4-ATPases from S. cerevisiae , A. thaliana and humans, and how they diverge from the P1, P2 and P5-ATPases which are also found in humans. The P4-ATPases are further classified into P4A, P4B and P4C-ATPases. The monomeric P4B-ATPases (dark teal) are evolutionarily older, while the heterodimeric P4A-ATPases (cyan) are the youngest, while the P4C-ATPases (teal) are an intermediary heterodimeric group. (C) Structural alignment of E1-ATP intermediates of Drs2 of the P4A-ATPase family with ATP1A1 (P2C), ATP2A2 (P2A), ATP7B (P1A), ATP13A2 (P5B) and Neo1 (P4B) indicates that the general structures are superimposable. The TM domain (light blue), P domain (green) and A domain (pink) generally exhibit similar positions. However, members such as the P1A-ATPases possess additional cytosolic domains for their specific transport functions. An initial sequence-based multiple sequence alignment (MSA) revealed that the level of sequence identity between the various subfamilies of human P-type ATPases was low ( Fig. 1A , 1B). P4-ATPases exhibited varying degrees of sequence conservation ranging from 27% to 75%, while they exhibited a sequence identity of ∼15-20% with other P-type ATPases. These residues were spread around both the transmembrane domains and the cytosolic domains. However, the structures of proteins of this family were highly superimposable ( Fig. 1C ). Additionally, the resolved structure of ScNeo1, a P4B-ATPase, revealed that structural alignment did not extrapolate to the primary sequence alignment with the much bigger P4A-ATPases. Consequently, the AlphaFold structures were used to perform a structure-based multiple sequence alignment to identify residues conserved in the P4-ATPase subfamily of proteins. Based on this, we identified residues which are conserved in all P4-ATPases ( Table 1 ), residues conserved in the whole P-type ATPase family of proteins ( Table 2 ), and residues conserved in P4-ATPases but diverge between P4A/C and P4B-ATPases ( Table 3 ). View this table: View inline View popup Table 1. Residues conserved in all P4-ATPases. View this table: View inline View popup Download powerpoint Table 2. Residues conserved in the whole P-type ATPase family. View this table: View inline View popup Table 3. Residues conserved in P4-ATPases but diverged between P4A/C and P4B-ATPases. Conservation color codes: green – highly conserved, light green – loosely conserved, white – not conserved. In addition to the divergence between P4A and P4B-ATPases, a higher frequency of substitutions was observed at several conserved residues in Dnf3, ATP8B3 and ALA1, suggesting functional impacts on these poorly understood substitutions. Moreover, specific substitutions were observed in ATP8B, ATP10 or ATP11 flippases in humans, speculatively due to specific substrate transport or other functions. By exploiting the fast-growing number of resolved structures of P4-ATPases, the identified residues were mapped out onto these structures to identify conserved regions in the protein structures ( Fig 2 ). The P-Domain contains the highest number of residues conserved in the whole P-type ATPase family located here, which are closer to the interface of the three cytosolic domains. Meanwhile, several P4A/B divergent residues were identified in the A and N-Domains. The A-P domain interfaces and the N-P domain interfaces are conserved across P-type ATPases, while the regions surrounding this A-N-P cavity differentiate P4-ATPases from other P-type ATPases. Download figure Open in new tab Figure 2. Distribution of conserved residues. Distribution of conserved residues in (A) cytosolic domains (B) transmembrane helices and their linkers, with the P-type (C), P4 (D) and P4A-B specific residues (E) mapped to the structure of Drs2 (PDB: 6PSX). Moving to the transmembrane domains, TM4-5 possess the few transmembrane residues conserved across P-type ATPases, while most of the transmembrane domains house residues conserved across P4-ATPases. However, TM5 and TM8 stand out due to their divergence between P4A and P4B-ATPases. 2.2. Aromatic pathways of the TM Domain Using Drs2 as the model and mapping these residues onto resolved cryo-EM structures, we have observed several conserved aromatic residues in the periphery of the protein, projecting toward the membrane ( Fig 3 ). Some of these residues were conserved among all P4-ATPases, while some exhibited variations between the P4A and P4B-ATPases. Of note, a series of phenylalanines and tyrosines were identified to be conserved in TM1 of the P4-ATPase family. These residues are oriented toward both the centre and periphery of the TM domain. In addition to this, several other aromatic residues were also found in this stretch, albeit not as conserved. Further, TM1-4 have several conserved phenylalanines and tyrosines, and TM5-10 has several tryptophans oriented into the lipid bilayer. Of note, some of these tryptophans are unique to the P4A-subclass. Download figure Open in new tab Figure 3. Aromatic Aisle and pathways of P4-ATPases. (A, B) A series of conserved aromatic residues were found to be conserved among P4-ATPases (coral). These were interspersed by other aromatic residues which are conserved to varying degrees. Several other aromatic residues were found to be conserved in both in P4-ATPases and P4A/B-ATPases, leading to several aromatic pathways in both P4A- and P4B-ATPases (C, D respectively). (PDB: 6PSX, 7RD6). Notably, the TM5 of the P4A-ATPases has a unique stretch of conserved aromatic residues, which exhibits semiconservative and non-conservative substitutions in the P4B-ATPases ( Figure 4A ). This region is proximal to the PISL motif and houses the YK(N/S) or HRG motif ( Figure 2B ). This Proline Pocket, specifically consisting of the VMHRG motif in P4B-ATPases, shows minute changes between different subfamilies of the P-type ATPase family, transporting different substrates ( Fig. 2A ). Of note, all the currently resolved structures of Neo1 show that the PISL motif is oriented completely away from the HRG motif, unlike in P4A ATPase structures, where it is oriented toward the YKN/S motif ( Fig. 2C ). Additionally, Drs2 N1050 , which has been a critical determinant of substrate specificity with a role in binding the phosphate group of phospholipids, exists as the much smaller alanine or, in some cases, serine in P4B-ATPases. At the nearby Drs2 N1050 , P4A-ATPases most commonly feature an asparagine residue, while P4B-ATPases possess a smaller residue at this position, typically alanine or serine. Download figure Open in new tab Figure 4. Adapting the Proline Pocket of P-type ATPases. (A, B) The PISL motif is a conserved motif in all P4-ATPases, which has a proline and leucine conserved in all P-type ATPases (blue) and a serine conserved in all P4-ATPases (coral). The YKN motif located proximally to this motif is required for binding substrates such as phosphatidylserine (black) (B), along with Drs2N1050 (purple). These residues are modified for a conserved VMHRG motif and Neo1A978 (purple) in P4B-ATPases. (C) Mapping the conserved residues of this pocket in Drs2 (PDB: 6PSX) also exhibits differences in the features of the proline in relation to this pocket in Neo1 (PDB: 7RD6). (D) On the other side of the Proline Pocket lies a conserved pathway that diverges between P4A and P4B-ATPases, whose function is currently unknown. Apart from this, we also identified a conserved pathway between TM5, TM7 and TM8 that diverges between the P4A and P4B-ATPases. This pathway includes the other residues from the VMHRG motif, including a strong salt bridge between R945 and E1065 in the P4B-ATPases. 2.3. Cytosolic domains The function of the cytosolic domain in P4-ATPases is similar to that of P-type ATPases in the way that they also involve a similar phosphoenzyme formation that is associated with changes in the transmembrane domain to transport substances. While many conserved residues in the P-type ATPase family are found in the cytosolic regions, they are far exceeded by the number of residues unique to the P4-ATPase subfamily of residues. Our analysis revealed several key conserved motifs in P-type ATPases that exhibit distinct but overlapping ligand-binding properties ( Fig 5 ). The previously identified DGET motif demonstrates broad ligand recognition: binding AlF 4 - , BeF 3- and VO 4 - in P4-ATPases or interacting with MgF 4 in P2B ATPases, in accordance with its role in dephosphorylation. The DKTG motif shows near-universal phosphate coordination, interacting with nearly all tested ligands except ATP in P4-ATPases and ADP in P1B ATPases. Similarly, the TGD motif was also found to interact with all tested ligands in all conditions. Download figure Open in new tab Figure 5. Conservation of motifs binding ATP and phosphate analogs in P-type ATPases. (A) Binding sites of ligands involved in binding ATP and phosphate analogs (PDB: 7OH7, 7OH5, 7PEM) and (B) their conservation in different P-type ATPase families. (C, D) While some of the motifs involved in binding these ligands are conserved across all P-type ATPases (blue), such as the DKTG, KGA, TGD and GDGxND motifs, others are unique to the P4-ATPase subfamily (coral). This includes motifs such as the SPDExALV and GxEGxQ. However, there are other motifs such as the CCR, PLQK, RTLC and RKRMS which exhibit a more mixed conservation pattern, where the residues interacting with these ligands are conserved across P-type ATPases, but the surrounding residues are unique to P4-ATPases, indicating that they might be uniquely positioning these motifs in P4-ATPases to allow other allosteric actions. The GxEGxQ motif uniquely binds AlF 4- and Mg 2+ (PDB: 8OX6) and ADP (PDB: 8OX5) in ATP8B1 as well as (F) AlF 4- in Dnf2 (PDB: 7KY9), although all three ligands were in proximity in all these structures. Adenine-specific recognition emerges as a major functional theme, with the SPDExALV motif primarily binding the adenine group across subfamilies, though with notable exceptions including AMPPCP binding in P1B and P5 and ATPgS interaction in P2C. Of note, it is also found to bind AlF 4 - in P4-ATPases, a feature not seen in other P-type subfamilies. Similarly, the FNSS motif engages the adenine group in most subfamilies, with the striking exception of P1B where it appears uninvolved in ATP binding. Similarly, the RKRMS motif also binds adenine containing molecules in most families, with the exception of ATPgS in P2C and AMPPCP in P1B. The KGA motif shows preferential binding to adenine-containing ligands (ATP, ACP, ADP) in most cases, though with exceptions in P1B (AMPPCP) and P5 (ATP, AMPPNP). The RTLC motif features a conserved arginine critical for binding ATP analogs and ADP across subfamilies, with AMPPCP binding in P1B again representing an exception. This divergence highlights subfamily-specific adaptations in nucleotide recognition, specifically the adenine group. Unique functional specializations are evident in certain motifs. The CCR motif demonstrates an unusual capacity to bind ALF in P4-ATPases, in addition to its interactions with AMPPCP and ADP. The PLQK and GDGxND motifs show remarkably broad ligand recognition but does not bind all these ligands in all these families. Most strikingly, the GxEGxQ motif appears exclusively in P4-ATPases where it shows highly selective binding to only ADP, ALF and Mg 2+ , especially for ATP8B1 ( Fig 5E , F). These findings collectively demonstrate both conserved principles and specialized adaptations in nucleotide recognition across P-type ATPase subfamilies. The data reveal how core structural motifs have evolved distinct ligand preferences while maintaining fundamental functions in ATPase activity. 3. DISCUSSION This study undertook a comprehensive sequence analysis of the P4-ATPase subfamily of phospholipid transporters, incorporating insights from the parent P-type ATPase family. While other studies have conducted sequence analyses of specific or all P4 and even all P-type ATPases ( Pedersen et al. 2012b ; Palmgren et al. 2019 ), recent structure-function studies from the Graham and other labs have highlighted the need for a comprehensive, systematic structure-based sequence analysis of P4-ATPases. Despite maintaining the general overall architecture and reaction cycle, the specific biochemical properties of the transmembrane and cytosolic domains differ between the various P-type ATPase subfamilies. This indicates that each subfamily possesses its own sensing and regulatory mechanisms to initiate ATP hydrolysis and substrate translocation. While even dephosphorylation exhibits some variation between the P1/2/3-ATPases and P4/5-ATPases, the residues involved in binding the ψ-phosphate of ATP are conserved across P-type ATPases, underscoring their critical role in the function of these proteins. Conversely, the ATP binding pocket shows several conserved motifs, exhibiting subfamily specific divergence among the different subfamilies of P-type ATPases in some sites. Most of the ATP binding residues are conserved in P-type ATPases but are flanked by residues specific to P4-ATPases, especially in the adenine recognition region. One exception is the SPDExALV motif that is highly conserved and unique in P4-ATPases, where as an equivalent but different motif exists in each subfamily of P-type ATPases. Of note, mutations to these motifs or proximal regions have often been associated with diseases in the P4 and other P-type ATPases. Mutations at the corresponding H1069 of the SEHPL motif in the P1B-ATPase ATP7B is a frequent cause of Wilson’s Disease, due to its impact on ATP binding and phosphorylation ( Tsivkovskii, Efremov, and Lutsenko 2003 ; Dmitriev et al. 2006 ). In the P4-ATPases, a mutation L538P immediately proximal to the SPDE motif of ATP8A2 has also been shown to cause Cerebellar Ataxia, Mental Retardation, and Disequilibrium Syndrome type 4 (CAMRQ4) ( Flannery et al. 2024 ). Apart from this, the Mg 2+ binding regions, DKTG and GDGxND remain conserved across P4 and P-type ATPases, although a GxEGxQ motif was also observed to be involved in ATP8B1 ( Dieudonné et al. 2023 ). This indicates that they could be involved in specific allosteric coupling to the TM domain in P4-ATPases. Further, the A and N-Domains also exhibits differences between the monomeric P4B-ATPases and the heterodimeric P4A/C ATPases, with some of these regions exhibiting stronger electrostatic interactions in P4B-ATPases compared to those in P4A-ATPases. Additionally, some of these regions have been implicated in binding the beta-subunit of P4A-ATPases, indicating that they could require stabilization. Previously, it was hypothesized that the extracellular loops 1, 4 and 5 were responsible for the unique monomeric existence of the P4B-ATPases. Their data also identified that the A and N-Domains were stable in the E1-ATP state ( Bai et al. 2021 ), unlike other P4-ATPases where the A and N-Domains are usually very flexible at this stage of the cell cycle ( Dieudonné et al. 2023 ; Hiraizumi et al. 2019 ; Nakanishi, Nishizawa, et al. 2020 ; Bai et al. 2020 ). Consequently, these substitutions in the A and N Domains could also contribute to the stability of P4-ATPases, potentially by stabilizing the dephosphorylation and nucleotide binding pockets. In the transmembrane domains, a critical substitution was identified near the substrate translocation pathway of P4B-ATPases. A TM6 asparagine (Drs2 N1050 ) plays a critical role in phospholipid occlusion. It coordinates a water molecule which is eventually replaced by the phospholipid. Mutagenesis of this residue critically impairs function in vitro and causes diseases such as CAMRQ ( Alsahli et al. 2018 ; Hiraizumi et al. 2019 ; Dieudonné et al. 2023 ; Nakanishi, Irie, et al. 2020 ). This residue exhibits a substitution to the much smaller alanine/serine/threonine in P4B-ATPases. The presence of a serine at this location could strengthen the water bonding network, slowing the process of lipid translocation, which in turn makes the outward open E1 state less transient and hence, stable. As such, this may contribute to the increased existence of the P4B-ATPases as monomers. The YKN/S motif of P4A-ATPases ( Figure 4 ) has been identified as a determinant of substrate selectivity in P4A ATPases ( Mikkelsen et al. 2019 ). When substrate binds to the proximal PISL motif, it transmits substrate binding induced changes to the P-Domain to induce dephosphorylation (Nakanishi, Irie, et al. 2020). This motif has also been implicated in binding and stabilization of the phosphate headgroup and ejection into the cytosolic leaflet ( Coleman et al. 2012 ). Mutations in this region have universally impaired substrate transport in P4-ATPases, including lethality in mutants of the essential Neo1 ( Palmgren et al. 2019 ). Consequently, the movement of water molecules and phospholipids through the P4B-ATPase transmembrane domains appears to have different characteristics, despite having similar determinants of substrate selectivity. Meanwhile, the roles of residues in the VMHRG-salt bridge pathway of TM5, 7 and 8 are completely unknown and require characterization in P4A and P4B-ATPases. Another conserved feature of the P4-ATPase transmembrane domain is the aromatic aisle on TM1. While Drs2 F230 has been implicated as part of the hydrophobic gate and regulates substrate specificity ( Mogensen et al. 2024 ; Hiraizumi et al. 2019 ; Nakanishi, Irie, et al. 2020), Drs2 F229 forms a smaller hydrophobic cluster that finetunes PS transport ( Mogensen et al. 2024 ). However, it is also worth noting that these residues form part of the cholesterol sensing CRAC ((L/V)-X1-5-Y-X1-5-(K/R)) and CARC motifs ((R/K)-X1-5-(F/Y/W)-X1-5-(L/V)), suggesting a larger role in sensing membrane composition and fluidity. The effects of these residues have been tested in the presence of conventional P4-ATPase substrate lipids such as PS, PE and GlcCer ( Mogensen et al. 2024 ; Roland et al. 2019 ). However, there exists a high frequency of aromatic residues in this transmembrane helix of P4-ATPases, some conserved across the whole family and some specific to individual members. Further, while we identified other CRAC and CARC motifs in different P4-ATPases the CRAC and CARC motifs of TM1 were the only ones conserved across different P4-ATPases. This suggests that membrane composition could allosterically control different P4-ATPases differently, while all of them are also regulated by a common mechanism involving TM1. Moreover, the higher incidence of tryptophans in TM5-10 could indicate a requirement for greater stability in this region, compared to TM1-4 which must undergo conformational changes coupled to the reaction cycle. In addition to these regions highlighted in the study, we also found that several key residues diverge between P4A- and P4B-ATPases. For instance, K224 and A226 (phenylalanines in P4B) were found in the middle of the electrostatic zipper of TM2 identified by the Molday group, while K442 and I449 (charged and hydrophobic residues reversed) were found on either side of the critical exit gate residue N445. While these residues have been implicated in substrate transport and CDC50 binding, the roles of most of these residues remain unknown. Experiments such as targeted alanine scanning mutagenesis in P4A and P4B-ATPases may determine the role of this conserved region in P4B-ATPases. Several key questions and future directions emerge in this study. First, further structural basis of monomeric P4B-ATPase stability and substrate translocation may be established through the generation of chimeric mutants of the extracellular, transmembrane and cytosolic domains based on residues/motifs identified in this study. This could also be complemented by coarse grained molecular dynamics simulations of the P4B-ATPase reaction cycle and atomistic simulations of water molecules or phospholipids through the P4B-ATPase TM domain. This will shed light on the energetic requirements of the reaction cycle that allow it to exist as a monomer, as well as how substitutions of critical motifs and residues near the substrate translocation pathway can support translocation of the same substrates as P4A-ATPases. Eventually the results will inform therapeutic strategies to rescue disorders such as progressive familial intrahepatic cholestasis (PFIC) which arise due to instability in P4A-ATPases. Secondly, the cytosolic domains of P4-ATPases possess many residues unique to this family in the cytosolic domains. While these have partly been attributed to the substrate-independent phosphorylation of P4-ATPases, most of these residues are not conserved in the P5-ATPases which also exhibit substrate independent phosphorylation. Consequently, motifs such as the SPDExALV, GxEGxQ motifs and P4-specific residues flanking the ATP binding motifs warrant further investigation through biochemical approaches such as protein folding experiments, phosphate analog-dependant TNP-AMP superfluorescence, phosphorylation assays, limited proteolysis and isolated cytosolic domain studies. This will shed more light on how nucleotide handling is specifically coupled to phospholipid transport in P4-ATPases. Finally, the mechanisms used by P4-ATPases to sense bilayer composition remain critically understudied. Systematic mutagenesis of the snorkelling aromatic residues along the sterol-sending motifs may be undertaken in reconstituted systems of varying phospholipid-cholesterol ratios. This would be complemented by computational, structural and cellular studies with varied cholesterol proportions to determine its impact on the conformational cycle of P4-ATPases. Such studies will shed more light on how P4-ATPases, which are embedded in a pool of their own substrates, are regulated by membrane composition and fluidity against constitutive activation. Such studies will shed light on how these flippases integrate dynamic signals from the membrane to maintain membrane asymmetry and regulate protein traffic. In conclusion, our comprehensive sequence and structural analyses provide a detailed structure-based map of sequence conservation and divergence across P4-ATPase subfamilies, identifying both shared functional elements and specialized adaptations. The findings highlight how P4-ATPases have evolved unique solutions to the challenges of phospholipid transport while maintaining the core P-type ATPase fold and reaction cycle. The conserved motifs and subfamily-specific variations identified here provide a framework for future mechanistic studies and structure-function analyses of these essential membrane transporters. Although we have attempted to identify regions of interest based on available structural and functional data, several residues identified in this study require further characterization to determine their role in P4-ATPases overall or among the specific P4A or P4B-ATPases. By integrating these findings with biochemical and structural approaches, we can advance our understanding of how P4-ATPases achieve lipid flipping and how their dysfunction contributes to human disease. 4. MATERIALS AND METHODS 4.1. Pairwise identity Canonical sequences for P-type ATPases ( Supplementary Table 1 ) were retrieved from UniProt (The UniProt Consortium 2025 ). Initial sequence analysis and pairwise identity for human P-type ATPases was conducted using ClustalOmega ( Sievers and Higgins 2018 ). The resulting tree and identity matrix was visualized as a heatmap using GraphPad Prism. 4.2. AlphaFold Structures Although structures have been resolved for various P-type ATPases, there exist several discrepancies (experimental technique, resolved state, completeness). Consequently, AlphaFold structures ( Abramson et al. 2024 ) (v4) were generated for all proteins mentioned in Supplementary Table 2 . 4.3. Structure based multiple sequence alignments The AlphaFold structures were submitted to PROMALS3D ( Pei, Kim, and Grishin 2008 ) to generated structure-based multiple sequence alignments. Due to the 20-structure limit imposed by the server, sMSAs of the individual families were generated. Further, given the P4B-ATPase family’s evolutionary closeness to the P-type ATPases, the characterized P4B-ATPases were aligned to each P-type ATPase subfamily to determine conserved residues of P4A, B, P4 and P-type ATPases. Further, these P4B-ATPases were compared against human, yeast and plant P4B-ATPases to identify residues which differed between the monomeric and heterodimeric subfamilies. The individual subfamily sMSAs were analyzed using JalView ( Waterhouse et al. 2009 ) to generate the Weblogos used in the figures. 4.4. Identification of ligand-binding residues Ligand-bearing structures of P-type ATPases were imported to ChimeraX ( Meng et al. 2023 ). The residues in contact with the ligand were identified using default parameters in the Contacts tool (VDW overlap >/= 0.4 Å). The union of ligand binding sites of for each ligand for all P4 and other P-type ATPases was used to generate the heatmap of ligand binding sites. 4.5. Sequence based multiple sequence alignment Canonical sequences of P4-ATPases were subjected to NCBI PSI-BLAST to identify 250 orthologs of each human and yeast P4-ATPase. The human and yeast P4-ATPases were limited to databases Mammalia and Fungi respectively. The conservation was initially curated by the PROMALS3D convention (≥7: highly conserved, ≥5: loosely conserved, <5 not conserved). The resulting hits were manually curated to remove partial, low quality and non-standard sequences, as listed in the tables. The resulting hits were submitted to ClustalOmega to generate a multiple sequence alignment. The conservation in this multiple sequence alignment was quantified using the ConSurf server. WebLogos were generated for these alignments using the WebLogo server ( Crooks et al. 2004 ). 5. AUTHOR CONTRIBUTIONS KVS and SASR carried out the initial multiple sequence alignment; KVS performed further structural and sequence analysis. KVS and JYL wrote the manuscript; JYL oversaw the project and acquired research fundings. All authors have read and agreed to the submitted version of the manuscript. SUPPLEMENTARY INFORMATION View this table: View inline View popup Supplementary Table 1. Sequences of P-Type ATPases used in this study. View this table: View inline View popup Download powerpoint Supplementary Table 2. Structures of P-Type ATPases used in this study. 6. ACKNOWLEDGEMENTS We thank the Protein Biophysics Core Facilities for the computational support and Ms. Fatemeh Rezaei for insightful discussions regarding cholesterol sensing motifs. This work was initiated with the support of the University of Ottawa Virtual Research Opportunities Internships to KVS and SASR. The project was funded by the Natural Sciences and Engineering Research Council Discovery Grants (RGPIN 2018-04070 & RGPIN-2025-04349) to JYL. Funder Information Declared Natural Sciences and Engineering Research Council, https://ror.org/01h531d29 , RGPIN 2018-04070 , RGPIN-2025-04349 7. 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Share Sequence Analysis of P4-ATPases Reveals the Structural Determinants for the Stable Monomeric P4B-ATPase Phospholipid Transporters Kadambari Vijay Sai , Sai Anvithaa Sundar Rajan , Jyh-Yeuan Lee bioRxiv 2025.07.01.662597; doi: https://doi.org/10.1101/2025.07.01.662597 Share This Article: Copy Citation Tools Sequence Analysis of P4-ATPases Reveals the Structural Determinants for the Stable Monomeric P4B-ATPase Phospholipid Transporters Kadambari Vijay Sai , Sai Anvithaa Sundar Rajan , Jyh-Yeuan Lee bioRxiv 2025.07.01.662597; doi: https://doi.org/10.1101/2025.07.01.662597 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Bioinformatics Subject Areas All Articles Animal Behavior and Cognition (7642) Biochemistry (17715) Bioengineering (13907) Bioinformatics (42003) Biophysics (21470) Cancer Biology (18624) Cell Biology (25533) Clinical Trials (138) Developmental Biology (13390) Ecology (19935) Epidemiology (2067) Evolutionary Biology (24356) Genetics (15617) Genomics (22529) Immunology (17753) Microbiology (40432) Molecular Biology (17200) Neuroscience (88681) Paleontology (667) Pathology (2840) Pharmacology and Toxicology (4828) Physiology (7653) Plant Biology (15161) Scientific Communication and Education (2046) Synthetic Biology (4304) Systems Biology (9826) Zoology (2271)