ABSTRACT
WalKR (YycFG) is the only essential two-component regulator in the human pathogen Staphylococcus aureus. WalKR regulates peptidoglycan synthesis, but this function alone appears not to explain its essentiality. To understand WalKR function we investigated a suppressor mutant that arose when WalKR activity was impaired; a single histidine to tryptophan substitution (H271Y) in the cytoplasmic Per-Arnt-Sim (PASCYT) domain of the histidine kinase WalK. Introduction of the WalKH271Y mutation into wild-type S. aureus activated the WalKR regulon. Structural analyses of the WalK PASCYT domain revealed a hitherto unknown metal binding site, in which a zinc ion (Zn2+) was tetrahedrally-coordinated by four amino acid residues including H271. The WallkH271Y mutation abrogated metal binding, increasing WalK kinase activity and WalR phosphorylation. Thus, Zn2+-binding negatively regulates WalKR activity. Identification of a metal ligand sensed by the WalKR system substantially expands our understanding of this critical S. aureus regulon.
Introduction
Staphylococcus aureus is a major human pathogen that causes a wide range of hospital- and community-acquired opportunistic infections1 Antibiotic-resistant strains (in particular methicillin-resistant S. aureus [MRSA]) are increasing in prevalence in both hospital and community settings. Resistance to last line agents such as vancomycin, linezolid and daptomycin is well described 23, casting doubt on their efficacy for the treatment of serious MRSA infections 45. In the context of limited treatment options, the social and financial burden posed by S. aureus related disease is now globally significant1
WalKR is a highly conserved two-component regulatory system (TCS) with features that are unique among low G+C Gram-positive bacteria6. Like other TCS, it comprises a multi-domain transmembrane sensor histidine kinase (WalK – HK) (Fig. 1a) and a response regulator (WaIR – RR). Notably, in S. aureus and closely related bacteria, the locus is essential, a characteristic that has made it a potential therapeutic target7. The WalR/WalK system (also called YycFG and VicRK) was first identified from temperature-sensitive mutants in Bacillus subtilis 8 and then S. aureus 9, with the system essential in both genera. Essentiality of WalKR has been inferred through both construction of strains containing an inducible WalKR 10,11, which were unable to grow in the absence of inducer, and the inability to delete the genes in Listeria monocytogenes or Enterococcus faecalis 12,13. In an impressive recent study, Villanueva et al14. deleted all 15 non-essential TCSs from two differents. aureus backgrounds and confirmed that WalKR is the only TCS strictly required for growth. Depletion of WalKR in B. subtilis produces a long-chain phenotype with the formation of multiple ‘ghost’ cells without cytoplasmic contents, which correlates with a reduction in colony forming units (CFU)15. In S. aureus, depletion of WalKR causes the same loss in viability, but without cellularlysis, although aberrant septum formation was reported 16. These observations suggest different mechanisms for the essentiality of WalKR in rod versus coccus shaped bacteria. However, this is not a uniform requirement as although the TCS is also found in streptococci, the WalK ortholog in this species is not essential and downstream regulators are absent, suggesting altered regulation. Further, WalK in streptococci contain only one transmembrane domain, which differentiates it from other cocci6.
S. aureus antibiotic resistance and clinical persistence are frequently linked to mutations in regulatory genes, in particular TCS. Among the S. aureus TCSs, mutations in loci such as vraRS, graRS and walKR a reassociated with the vancomycin intermediate resistant S. aureus (VISA) phenotype 17-19. Notably, mutations in WalKR are a critical contributor toward this phenotype with numerous clinical VISA strains possessing walKR mutations 20. Induction of characteristic VISA phenotypes (thickened cell wall, reduced autolytic activity and reduced virulence) can arise from mutations as simple as a single nucleotide change in either walK or walR 21-23.
Despite the central role of the TCS in bacterial viability, the physiological and/or mechanical signal sensed by WalK is unknown. In B. subtilis, where it has been more extensively studied, WalK localises to the division septum and interacts with proteins of the divisome 24. B. subtilis WalR positively regulates several autolysins involved in peptidoglycan metabolism and represses inhibitors of these enzymes 25. Thus, WalKR has been inferred to link cell division with cell wall homeostasis 624. Nevertheless, its role in S. aureus appears to be distinct. Although S. aureus WalR does control autolysin expression 16,26, this function does not explain the essentiality of the system as expression of genes encoding lytic transglycosylases or amidases in a WalKR-depleted strain do not restore cell viability 27. Further, the membrane associated regulators YycHI act as an activator of WalK function in S. aureus 28, which contrasts with B. subtilis where they serve to repress the system 29.
Sequence variation provides one basis for the apparent differences in WalKR function between S. aureus and B. subtilis. The WalK alleles share only 45% amino acid identity, with the majority of the variation concentrated in the extracellular region and a cytoplasmic Per-Arnt Sim (PAS) domain (Fig. la). Although these regions have low sequence conservation, PAS domains are known to adopt a conserved tertiary fold, where they facilitate sensory perception via ligand interaction enabling signal transduction. In HKs that contain PAS domains, known ligands include heme, flavin mononucleotide and di/tricarboxylic acids30. In B. subtilis, the cytoplasmic PAS domain is essential for WalK localisation to the division septum 24. Recently, S. aureus WalK was also shown via a GFP- fusion to be to be preferentially located to the division septum. The role of the cytoplasmic PAS domain in septum targeting was not examined31.
Here, we screened S. aureus yycHI deletion mutants and identified a novel WalK suppressor mutation (H271Y) located in the cytoplasmic PAS domain. Structural and functional analyses of WalK revealed that this residue is a critical component of a cytoplasmic metal binding site that directly influenced HK activity. This metalloregulatory site was observed to bind zinc (Zn2+) in vitro, and this was abrogated by the H271Y mutation resulting in increased WalK autophosphorylation. These observations were corroborated in vivo through the construction of a metal-binding site mutant at the native locus with subsequent activation of WalKR-associated phenotypes and increased WalR phosphorylation in vivo. Molecular modelling of WalK in Zn2+-free and Zn2+-bound states suggests that metal occupancy influences conformational changes associated with the cytoplasmic domains, thus providing a plausible mechanism of activity modulation. To our knowledge, this is the first description of metal binding to the cytoplasmic PAS domain influencing the activity of a histidine kinase.
RESULTS
Mapping of a suppressor mutation to walK by genome sequencing
YycH and Yycl are membrane-associated accessory proteins that have been shown to directly interact with WalK and positively regulate WalKR function 28. We constructed an S. aureus yycHI deletion mutant (ΔyycHi) in USA300 strain NRS384 and observed colony sectoring after 2 days growth at 37°C on BHI agar (Fig. lb), suggesting the development of suppressor (compensatory) mutations. We purified the two colony morphotypes and performed whole genome sequencing. Aligning the sequence reads to the closed NRS384 genome revealed only a single point mutation in addition to the engineered yycHI deletion. The mutation occurred in the cytoplasmic PAS domain of WalK, wherein histidine 271 was predicted to be replaced by tyrosine (WalKH271Y). Despite the different chemical profiles of the two side chains, i.e. positively charged versus slightly polar, the two residues have similar steric bulk.
Generation of a walkH271Y site-directed mutant
We next sought to investigate the impact of the WalKH271Y substitution by introducing the mutation into wild-type NRS384 by allelic exchange. The resulting unmarked mutant was analysed by genome sequencing to exclude the occurrence of secondary site mutations, as was previously observed to occur in backgrounds involving mutagenesis of WalK 22,32. Here, only the nucleotides targeted by the allelic exchange of walK differed between wild-type walK and the walKH271Y mutant. The allelic exchange procedure was then repeated to convert the walK H271Y allele back to wild-type with the introduction of a silent Pst\ restriction site to mark the revertant (walKH271Y-COMP). By comparison with the wild-type, the wak H271Y mutant formed smaller colonies on sheep blood agar with reduced pigmentation, but produced a slightly larger zone of hemolysis (Fig. lc). Deletion of yycHI resulted in reduced hemolysis. However, hemolysis was elevated upon the introduction of the walK H271Y allele into the ΔyycHI background (Fig. lc). Intriguingly, following growth in TSB at 37°C, the WalKH271Ystrain did not exhibit a lag phase upon inoculation into fresh medium (Fig. 2a). Nonetheless, at 2 h post inoculation the growth rate was significantly reduced in comparison with the wild-type and complemented strain. The maximal doubling rate was 33 min for the WalKH271Y mutant versus 23 min for the wild-type and complemented strains. Further, the WalKH271Y mutant strain had a reduced final optical density at 600 nm (OD600) compared to the wild-type, although this equated to identical CFU counts (Fig. 2a).
The exoproteome and Atl activity is altered in the WalKH271Y mutant strain
Atl is a major peptidoglycan hydrolase produced by S. aureus that is positively controlled by WalKR 16,26. It is involved in daughter cell separation and also plays roles in primary attachment during biofilm formation and in the secretion of moonlighting proteins 33. Here, we analysed the secretion of proteins during exponential and stationary phase growth from the wild-type and the mutant WalKH271Y, WalKH271Y-COMP and Δ atl strains. We observed an increase in protein abundance in the WalKH271Ymutant in both exponential and stationary phases, by comparison to the wild-type levels observed in the walKH271Y-COMP strain. In contrast, secreted protein levels were reduced in the Δatlstrain compared to the wild-type (Fig. 2b). We then directly assessed Atl exoproteome by zymogram analysis, using Micrococcus luteus cells as a substrate. The Δatl strain activity in the displayed no visible lytic activity in contrast to the other strains, indicating that the majority of lysis is attributable to Atl34 (Fig. 2c). In stationary phase, there was an apparent increase in the amidase (63 kDa) and glucosaminidase (53 kDa) activity in samples from the walKH271Y mutant. These data indicate that the WalKH271Y mutation results in increased production and/or activity of Atl.
Increased lysostaphin and vancomycin sensitivity of the WalKH271Y mutant
WalKR regulates autolysis and directly influences sensitivity to vancomycin and lysostaphin 16,22,23,32.Here, we used lysostaphin and vancomycin sensitivity as indirect measures of WalKR activity. After a 90 min exposure to 0.2 μg/mL of lysostaphin, NRS384 exhibited a 0.5-logio10 reduction in cell viability, while the WalKH271Y mutant showed a further 0.5-logio10 increase in sensitivity. Complementation returned lysostaphin sensitivity to wild-type levels (Fig. 2d). In contrast, the ΔyycHI mutant displayed increased lysostaphin resistance compared to the wild-type, consistent with YycHI positively regulating WalKR. Therefore, the knockout of yycHI leads to reduced WalKR-controlled autolytic activity (Fig. 2d) 28. The compensatory WalKH271Y mutation in the ΔyycHI background did not fully restore lysostaphin sensitivity to wild-type levels. However, increased sensitivity to lysostaphin was observed at a higher concentration (1 μg/mL) with the ΔyycHI walkH271Y strain 2- log10 less viable than the ΔyycHI strain (Fig. 2d) with the wild-type below the limit of detection. Building on this framework, we then analysed a walKG223D mutant, which was previously shown to have reduced autophosphorylation/phosphotransfer between WalK and WalR 22,23. Here, this mutant showed increased resistance to lysostaphin, similar to that observed for the ΔyycHI mutant (Fig. 2d). We then analysed the impact of the mutations on vancomycin resistance, using antibiotic gradient plates (Fig. 2e). The wild-type, WalKH271Y-COMP and Δatl strains all showed similar levels of resistance. However, the AyycHI and WalK mutant strains showed increased resistance to vancomycin, while the WalKH271Y strain exhibited increased sensitivity compared to the wild-type. Introduction of WalKH271Yin the ΔyycHI background restored sensitivity to wild-type levels (Fig. 2e). These findings were consistent with Vitek 2 and Etest analyses of the strains (Fig. 2e). Collectively, these results indicate that the WalKH271Y mutation activates the WalKR system, resulting in increased sensitivity to lysostaphin and vancomycin.
Structure of the WalK PAS domain
To gain further insight into the impact of the WalKH271Y mutation, we determined a high-resolution structure of the cytoplasmic PAS domain. Domain boundaries for WalK-PAS were defined, based on limited proteolysis, as valine 251 to arginine 376. This PAS domain sequence (WalK-PASFULL) was cloned and heterologously expressed as a fusion protein with an N-terminal glutathione S-transferase (GST) tag and a thrombin cleavage site. WalK- PASFULL was purified by affinity chromatography, then the affinity tag was removed prior to crystallisation of the PAS domain (Suppl. Fig. 1). A selenomethionine derivative was also generated to aid in phasing the diffraction data (Suppl. Fig. 2). The WalK-PASFULL structure was solved to 2.0Å, although a lack of density for the N- and C- termini precluded modelling residues 251-262 and 370-376, respectively. Density was also absent for residues 335-338, which represents a disordered loop in WalK-PASFULL. Details of the diffraction data and structure statistics are summarised in Table SI.
The WalK-PASFULL structure has a typical PAS domain fold 35 comprising five antiparallel β-strands and four α-helices, with connecting loops between the helices and β-strands (Fig. 3a,b). The N- terminal region of the structure is composed of two short β-strands connected by a small loop. The remainder of the N-terminal region is comprised of four short helices connected by two large loops. The C-terminal portion of the PAS domain predominantly consists of a short β-strand followed by two larger antiparallel β-strands connected by a loop. Analysis of the surface electrostatic potential showed an uneven charge distribution on the WalK-PASFULL surface (Fig. 3c). In contrast to other PAS domains, a well-defined and continuous electron density was observed for a single zinc atom coordinated by a hitherto unknown metal-binding site (Fig. 3d). Notably, the Zn2+-binding site resides on the surface of the WalK-PASFULL domain, with access for Zn2+ from the surrounding solvent. The metal binding site comprises a single Zn2+ ion bound by the atoms Nδ1 from His271, 0δ1 from Asp274, Nδ1 from His364, and Oe2 from Glu368 in a slightly distorted tetrahedral coordination geometry (Fig. 3d, Suppl. Fig. 3a). The observed bond lengths are 2.20Å, 2.23Å, 2.23Å, and 2.24Å, respectively, and are typical for protein coordination of a Zn2+ ion 36. The Zn2+-binding site, along with the rest of the exposed region, exhibits a negatively-charged surface, while the remainder of the WalK-PASFULL surface is relatively neutral. Although the N-terminal region is well ordered in the structure, the mobility of this region may increase in the absence of the Zn2+ ion and the stabilising interactions conferred by His271 or Asp274.
Analysis of WalK-PAS domain interaction with Zn2+
Further examination of the interaction of the PAS domain with Zn2+was performed using a truncation mutant (WalK-PASTRUNC) in which the unstructured regions (15 residues deleted from the N-terminus and 6 residues from the C-terminus) were removed. The high-resolution structure of WalK-PASTRUNC was solved to 2.1 Å (Table S2). Comparison of the WalK-PASFULL and WalK-PASTRUNC structures revealed nearly identical PAS domain folds (Suppl. Fig. 3b,c), with a calculated RMSD of 0.48Å.
To investigate the Zn2+ binding capability of WalK-PAS, we conducted Zn2+ binding assays with both WalK-PASTRUNC and WalK-PASTRUNC H271Y, followed by direct metal quantitation by inductively coupled plasma-mass spectrometry (ICP-MS). Zinc binding analyses of WalK-PASTRUNC revealed a metakprotein molar ratio of ˜0.3:1, suggesting that WalK-PAS was ˜30% Zn2+-bound at any given time. By contrast, no Zn2+ binding was observed for WalK-PASTRUNCH271Y. Taken together, these data indicate that the H271Y mutation abolishes Zn2+ binding by the WalK PAS domain, supporting the inference that metal binding at this site influences WalK activity. Isothermal titration calorimetry (ITC) revealed that WalK-PASTRUNC binds a single Zn2+ ion with Kd ˜340 nM, indicating a moderate affinity interaction, consistent the ICP-MS analysis. Analysis of WalK-PASTRUNC H271Y by ITC did not detect Zn2+ binding, consistent with the ICP-MS result. These data suggest that Zn2+ is unlikely to serve a structural role in the WalK-PAS domain, since structural Zn2+ sites in proteins are typically formed by coordination spheres comprising Cys4 or Cys2-His-Cys ligands and they are associated with much higher binding affinities for Zn2+ (100 nM – 100 pM)36. By contrast, Glu and Asp residues are infrequently found in structural Zn2+ sites, with a recent analysis of NCBI Protein Data Bank noting a prevalence of < 6.0% for relevant protein structures 36. Collectively, these data, in combination with the phenotypic observations for the walk H271Y mutant, strongly suggest that the metal binding site in WalK-PAS serves a regulatory role than a structural function.
PAS-domain Zn2+-binding regulates WalK autophosphorylation
To elucidate the regulatory role of metal binding in the WalK-PAS domain, we examined the impact of the H271Y mutation on WalK autophosphorylation activity. Measurements were performed using the cytoplasmic domains of WalK208608 (WalKCYT), which has previously been studied using autophosphorylation assays 10,23. Here, we observed that the H271Y mutation (WalKCYTH271Y) resulted in a ˜50% increase in autophosphorylation, by comparison to WalKCYT (Fig. 4a). Hence, these data indicate that Zn2+- binding by the PAS domain negatively regulates the autophosphorylation activity of WalK in vitro, consistent with the altered WalK activity observed by in vivo phenotypic analyses.
To complement the in vitro findings, we investigated the in vivo phosphorylation status of WaIR. We first established the detection efficiency for the liable aspartic acid phosphorylation on residue D53 of WaIR. This was achieved by constructing two WalR variants into the tetracycline inducible plasmid pRABll, containing either: (i) native WalR with a 3xFLAG tag on the C-terminus (WalR- FLAG); or (ii) a mutant variant of WalR-FLAG, wherein mutation of D53 to an alanine (D53A). The D53A mutation abolishes the potential for phosphorylation at this site. The above plasmids along with empty pRABll were transformed into the 5. aureus NRS384 wild-type, WalKH271Y, Δstpl (serine threonine phosphatase) and ΔpknB (serine threonine kinase). The latter two strains were included to differentiate the site of phosphorylation. PknB has been shown to phosphorylate WalR at residue T10137 while in B. subtilis, the deletion of Stpl led to increased WalR phosphorylation at the site equivalent to WalR T10138. Here, we observed that D53 phosphorylation on WalR was absent in the WalRD53 FLAG (Fig. 4b). Deletion of ΔPknB or Stpl did not alter D53 phosphorylation, indicating that WalK was the dominant contributor. Further, our analyses did not detect a second site of phosphorylation under these conditions, suggesting that T101 was not being phosphorylated or detected under the conditions tested. Increased phosphorylation of WalR was observed in the WalKH271Y background (Fig. 4b), consistent with the autophosphorylation results (Fig. 4a). Building on this framework, we examined D53 phosphorylation in the native context using chromosomally-tagged strains (WaIR) in either wild-type or WalK S. aureus. The ability to modify the chromosomal copy of walR showed that the FLAG tag did not dramatically alter WalR activity. When analysed throughout growth, there was a striking increase in the phosphorylation of WalR in the WalKH271Y strain over the wild-type from mid-log phase onwards, with the difference exaggerated as growth progressed into late log/early stationary phase (Fig. 4c,d). These findings are consistent with the WalK autophosphorylation assay (Fig. 4a) and show that the negative regulation of WalKR imposed by Zn2+ binding to the cytoplasmic PAS domain is important in dampening the response of WalK as the cells transition out of active growth.
Conservation of metal binding sites in WalK is restricted within the Firmicutes
We next examined the potential conservation of the novel WalK PASCYT domain metal binding site across low G+C Gram-positive bacteria. Alignment of a selection of WalK proteins from different genera with the S. aureus WalK reference sequence revealed that H271 is conserved among the coagulase positive/coagulase negative staphylococci and enterococci, but not in streptococci or listeria, where it is replaced by a tyrosine residue (Fig. 5a). A Y271 residue was also present in two out of the five bacilli examined, including the well-studied strain, B. subtilis 168. Notably, the three additional metal coordinating residues were only conserved among staphylococci and enterococci, with all other genera having at least one deviation from the S. aureus consensus. These results suggest that conservation of metal binding by WalK in staphylococci and enterococci might enable additional regulatory control of the essential WalKR TCS. We next examined the structural alignment of the WalK-PAS domains of S. aureus with that from Streptococcus mutans (WalK SM); the latter natively encodes the Y271 substitution and was crystallized in the context of the complete cytoplasmic domain 39 (Fig. 5b). Although the two PASCYT domains align with a relatively large RMSD of 2.31 Å, indicating significant structural differences, the largest deviations occur in the regions comprising the Zn2+-binding site of thes. aureus PASCYT domain. In S. mutans the WalKCYTSM domain forms a leucine zipper dimeric interface with an adjacent monomer. Metal binding by S. aureus WalK-PASCYT is predicted to preclude formation of such an interface. The Zn2+ atom would be positioned near the center of the structure, resulting in steric clashes at the dimeric PASCYT domain interface that would likely impede WalK dimer interactions (Fig. 5c).
Metal-induced conformational changes in WalK
To further understand the structural consequences of WalK Zn2+-binding and impact on kinase activity, we employed molecular dynamics (MD). MD simulations indicated that Zn2+-binding directly influences the relative positioning of the PAS and catalytic (CAT) domains. In the absence of Zn2+, the dihedral angle between the PASCYT and CAT domains in each monomer was ˜136° when viewed down the central axis of the kinase (measured as the dihedral angle between Cα atoms of residues 288, 271, 369 and 569, averaged between the two chains), while the average distance between the upper and a and averaged between the two chains) was 21.6 Å (Fig. 6a). In the presence of Zn2+, the relative dihedral angle increased to 175° while the intra-helical distance decreased to an average of 12.3 Å (Fig. 6b). The binding of the metal ion also stabilizes the PAS domain fold by bringing the N- and C- terminal regions into closer proximity. This is particularly evident for the N-terminal H271 (Tyr in WalK-PASSM) and the C-terminal Glu368 residues. Collectively, our structural analyses suggest that metal binding in the PAS domain results in significant conformational changes. These metal-induced structural rearrangements provide a mechanistic basis for how conformational changes arise in WalK to negatively regulate its autokinase activity and subsequent signal transduction to the response regulator, WaIR.
DISCUSSION
In this study, we provide the first evidence of a specific ligand for the WalKR system, opening new avenues to understand the function and essentiality of this regulon. The S. aureus WalKR two-component system was identified in the late 1990s, but the ligand(s) sensed by this histidine kinase and subsequent mechanisms of activation have remained elusive. One proposed model for WalK sensing has been through the recognition of the D-Ala-D-Ala moiety of Lipid II via the extra-cytoplasmic PAS domain 6. This molecule would be abundant at the site of septation during exponential growth, but become limiting upon the cessation of cellular replication. Although this scenario provides a link between activation of autolysin production, via phosphorylation of WaIR, with division septum localisation of WalK, there remains a paucity of experimental evidence to support this model 40. Here, we have shown that the presence of a metal binding site within the PASCYT domain of WalK directly influences the activation status of the protein. Abrogation of in vitro Zn2+-binding capacity of WalK increased the autophosphorylation of WalK and in vivo phosphotransfer to WaIR. There are several key examples of regulation of histidine kinase activity via PAS domain ligand-binding, such as oxygen sensing by Bradyrhizobium japonicum FixL, wherein heme binding by the cytoplasmic PAS domain regulates nitrogen fixation under reduced oxygen tensions 35; oxygen sensing by Staphylococcus carnosus NreB, in which the PASCYT domain contains an oxygen-labile iron-sulfur cluster 41; and redox status sensing by Azotobacter vinelandii NifL, where the PASCYT domain binds nicotinamide adenine dinucleotide 42. Direct metal ion binding has previously been observed in extracellular PAS domains, such as PhoQ. from Salmonella typhimurium, which senses the cations Ca2+ and Mg2+43. However, to the best of our knowledge, metal binding by a cytoplasmic PAS domain has not previously been reported and appears to be a highly restricted attribute among the staphylococci and enterococci, despite WalK being conserved among the Firmicutes. For instance, the WalK PASCYT domain in Streptococcus mutans has a naturally occurring tyrosine at residue 271 and structural analysis shows no evidence of metal binding39.
Recapitulation of this mutation in vivo resulted in phenotypes associated with activation of WalK (e.g. sensitivity to lysostaphin and vancomycin, increased hemolysis and activity/production of the major autolysin Atl) along with the loss of lag phase upon inoculation into fresh media. This latter phenotype would be consistent with the requirement for accumulation of a ligand sensed by the extracellular PAS domain leading to the activation of WalKR, and subsequent autolysin production. By contrast with B. subtilis, YycHI are activators of the S. aureus WalK system 28,29. Consequently, the metal-dependent regulation of the WalK-PAS domain in S. aureus may serve as a dynamic constraint on kinase activity. Additional levels of regulation of the WalKR operon have also been identified. These include a second site of phosphorylation, residue T101 on WaIR, by the serine threonine kinase known as Stkl or PknB 37, and the interaction of SpdC (previously called lysostaphin resistance factor A – lyrA) 44 with WalK, which negatively regulate genes under the control of WaIR45. Both PknB and SpdC are localised to the division septum similar to WalK 31,37, highlighting the complexity of this regulatory axis. The establishment of phos-tag acrylamide to analyse the phosphorylation status of WalR in vivo is a powerful tool to investigate the regulation of this essential system (Fig. 4).
The PASCYT domain of S. aureus WalK binds Zn2+ with only moderate affinity, suggesting a regulatory role for metal binding rather than an obligate structural function. In this manner, it is possible that this site only has transient interactions with Zn2+ thereby facilitating a continuum of WalK activation states rather than serving as a binary switch. Disruption of PASCYT domain dimerization upon metal binding could impact WalK activity in a similar manner to the L100R mutation in Streptococcus pneumoniae 46. The L100R mutation destabilizes S. pneumoniae PAS dimerization, leading to a loss of autophosphorylation activity. Our structural analyses suggest that Zn2+ binding induces large conformational changes that alter the relative positions of the PAScyT and catalytic domains are induced by Zn2+-binding. These observations provide a plausible mechanistic basis for the reduction in WalK function, although the precise mechanism remains to be elucidated.
Intriguingly, although the amino acid sequence differences between WalK from the staphylococci and enterococci compared to other Firmicutes are small, these differences appear to have major functional consequences. This may suggest differing or additional regulatory roles for WalK beyond peptidoglycan biosynthesis in these genera. Potential regulatory roles for WalK could include contributing to maintenance of intracellular metal homeostasis. Sensing of intracellular Zn2+ abundance by WalK, could influence the import and/or efflux of cytoplasmic Zn2+, via WalR mediated phosphorylation. However, transcriptome studies of S. aureus WalKR mutants commonly highlight genes and pathways involved in nucleotide metabolism, and not those associated with metal ion transport 22,26. Nonetheless, indirect metal-dependent regulation might influence such pathways. One potential mechanism would be via Stpl, a Mn2+-dependent phosphatase, and its cognate kinase PknB, which interacts with WalKR and influences its activity. Further insight into the role of metal coordination in regulating this histidine kinase is necessary to understand the essentially of the system in S. aureus.
METHODS
Strains, primers, reagents and media
Bacterial strains/plasmids and primers (IDT) used are described in Table S2 and Table S3, respectively. S. aureus were routinely grown on Brain Heart Infusion (BHI) agar (Difco) or in Tryptone Soy Broth (TSB) (Oxiod) at 37°C with shaking at 200 rpm. For the selection of pIMAY-Z containing strains, BHI agar was supplemented with 10 μg/ml chloramphenicol and 100 μg/ml X-gal (BHIA-CX). For protein expression, Terrific Broth (TB) was used (10 g/L tryptone, 24 g/L yeast extract, 10 g/L glucose, 0.17 M KH2P04and 0.72 M K2HP04). Restriction enzymes, Phusion DNA polymerase and T4 DNA ligase were purchased from New England Biolabs. Phire Hotstart II (for colony PCR) was purchased from Thermo Fisher. Genomic DNA from S. aureus was isolated from 1ml of an overnight culture (DNeasy Blood and Tissue Kit—Qiagen) pretreated with 100μg of lysostaphin (Sigma cat. no. L7386). Lysostaphin sensitivity assays were performed as described32.
S. aureus site-directed mutagenesis by allelic exchange
The walKH271Y mutation was recombined into the chromosomal copy of walK in the USA300 background (NRS384) by allelic exchange. The region encompassing walK was amplified by SOE-PCR to introduce a neutral change in the third nucleotide of codon 270 and the first nucleotide in codon 271 with primer set IM7/IM8/IM9/IM10 (Table S4). The resultant 2.7 kb product was recombined into pIMAY-Z by the seamless ligation cloning extract (SLiCE) method 32,47 and transformed into E. coli IM08B 48. The sequence confirmed construct was extracted from E. coli, ethanol precipitated and transformed into electrocompetent NRS384 49. Allelic exchange was performed as described 48, with the mutation screened using primer pair IM11/IM12 with an annealing temperature of 65°C. Reversion of the walKH271Y mutation to walK C0MP-Pstl was achieved through allelic exchange in NRS384 walK H271Y. A marked walK allele was constructed through the introduction of a silent Pst\ site (walK nucleotide 1302, A to G) into walK by SOE-PCR with primer set IM7/IM58/IM59/IM10. A 3xFLAG tag was introduced onto the C-terminus of WalR by SOE-PCR with primers IM31/IM108/IM109/IM40. Deletion of genes or insertion of a FLAG tag was performed by SOE-PCR with the product cloned into pIMAY-Z by SLiCE with the following primer sets: Δatl gene (IM96/IM97/IM98/IM99: entire gene), ΔyycHI (IM54/IM78/IM79/IM75: from codon 5 of yycH to the stop codon of yycl), Δstpl (IM251/IM252/IM253/IM254: from the start codon leaving the last 8 amino acids), ΔpknB (IM255/IM256/IM257/IM258: entire gene) or walRFLAG (IM31/1M108/1M109/1M40). The walK2230 mutation was directly amplified by PCR on JKD6008 genomic DNA with IM7/IM10 (Table S4). The purified pIMAY-Z construct was transformed into NRS384 after passage through IM08B (Table S4). Allelic exchange was performed as described above. To screen for the walkJ:LAG, IM111/IM181 was used to identify the insertion. For WalKG223D after allelic exchange, white colonies were screened for decreased sensitivity to vancomycin. Genome sequencing and analysis of the isolates was conducted as described previously32.
Construction of anhydrotetracycline (ATc) inducible WalR-FLAG
The WalRFLAG gene was PCR amplified from NRS384 walRFLAG genomic DNA with primers IM280/IM305 (wild-type) or by SOE-PCR with primers IM280/1M278/1M279/1M305 to introduce the walRD53A mutation. The products were digested with Kpnl/BamHI and ligated into Kpnl/Bglll digested pRAB11. Ligations were transformed into IM08B to produce either pRAB11 walR FLAG or Prab11 walR D53AFLAG. Plasmids (pRAB11, pRAB11 walR FLAG or pRAB11 walR D53AFLAG) were transformed into NRS384, NRS384 walR H271Y, NRS384 Δ stpl or NRS384 Δ pknB and selected on BHIA- CX at 37°C.
Antibiotic resistance profiling
For vancomycin gradient assays were performed as described except a 0 to 2 μg/ml gradient was used 50. After 24 h incubation at 37°C, plates were imaged. With the exception of Vitek 2 and Etests, all antibiotic susceptibility testing of strains was performed in triplicate. To access the susceptibility to a range of antibiotics, strains were tested on a Vitek 2 Gram Positive ID card (AST-P612; Biomerieux) as per manufacturer’s instructions. Extended glycopeptide susceptibilities were determined with vancomycin Etest strips (Biomerieux) using a 2.0 McFarland inoculum on thick BHI agar and incubation of 48 h at 37°C.
Supernatant protein precipitation and SDS-PAGE analysis
Supernatant proteins from overnight culture or OD600 = 0.8 TSB cultures were precipitated with 10% trichloroacetic acid at 4°C for 1 h. Pellets were washed with 1 ml of ice-cold acetone and air dried at room temperature. Precipitated proteins were resuspended in 2x SDS-PAGE loading buffer and prior to loading the samples were equilibrated with an equal volume of 1 M Tris.CI [8]. Zymogram analysis of supernatant proteins was conducted as described by Monk etal.51.
Cloning, expression and purification of the GST-WalK PAS domain in E. coli
WalK-PASFULL (residues Val251-Arg376) was PCR amplified from NRS384 genomic DNA with oligonucleotides WalK-PASFULL-F/WalK-PASFULL-R and cloned into pGEX-2T (BamH\/EcoR\) to yield a N-terminally tagged GST-PAS construct. For WalK-PASTRUNCand WalK-PASTRUNCH271Y (residues Asp266 to Glu371), codon-optimised genes were ordered from GeneArt, and these were sub-cloned as BamH\/EcoR\ fragments into pGEX-2T yielding pGEX-2T(WalK-PASTRUNC) and pGEX-2T(WalK-PASTRUNC H271Y). Overnight cultures of BL21 containing the different GST constructs were diluted 1:100 into 2 L of TB at 37°C at 180 rpm and grown to OD600 of 0.8. The culture was then induced with 0.4 mM IPTG and shifted to 16°C at 120 rpm for 10 h (final OD600 of ˜3). The cells were harvested by centrifugation at 5,000 g for 15 min at 4°C. The pellet was resuspended in 50 ml of GST lysis buffer (50 mM Tris.CI [8.0], 500 mM NaCI, 1 mM EDTA) containing 1 mg/ml lysozyme plus lU/ml DNase A and incubated for 30 min on ice. Cells were lysed at 40 kpsi in a cell disruptor (Constant Systems), then cell debris was removed by centrifugation at 39,000 g for 30 min at 4°C. The supernatant was collected and passed through GST-affinity resin in a gravity-flow column (Bio-Rad). The resin was then washed with two column volumes of GST lysis buffer followed by equilibration with 1 column volume of TCB (20 mM Tris [8.0], 500 mM NaCI, 1 mM CaCl2). WalK-PAS was eluted by on-column digestion with 20 ml. of TCB containing 200 U of thrombin. Thrombin digestion was carried out by incubating the column at room temperature for 30 min followed by 30 min at 37°C and 60 min at room temperature. The liberated PAS domain was purified by size exclusion chromatography using a Superdex 75 column. Purified PAS domains were concentrated using an Amicon centrifugal concentrator with a 10-kDa size cut-off. Protein purity was always greater than 95% as determined by SDS-PAGE with a yield of about 20 mg/L.
Expression and purification of selenomethionine-labelled WalK-PAS domain
A single colony of BL21 + pGEX-2T(WalK-PASFULL) was picked and used to inoculate 10 mLof pre-warmed L-broth (LB) at 37°C for 6 h (180 rpm). Minimal media (100 mL) was inoculated with 500 μL of this pre-culture. Cells were grown overnight at 37°C with shaking at 180 rpm, then 25 mL of culture was added to 2 L minimal medium and incubated at 30°C. After further growth to OD600 ˜ 0.7, amino acid stock solution containing selenomethionine was added, then cells were grown for 1 h before induction with 0.4 mM IPTG. Cells were harvested by centrifugation at 6,000 g for 20 min at 4°C and the resulting cell pellet stored at – 80° C. The SeMet-labelled WalK-PASFULL was purified as described above. Labelling was confirmed by peptide mapping through matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS). A total of 10 μ g of purified protein was subjected to digestion for 16 h with 125 ng porcine trypsin (MS-grade, Promega) in 200 mM tetraetyl ammonium bicarbonate [8.0] containing 10 % acetonitrile. The peptide digest was mixed in a 1:1 v/v using solution of α-cyano-4 hydroxycinamic and matrix (5 mg/ml in 50 % acetonitrile in 0.1 % trifluoroacetic acid), spotted directly onto stainless steel MALDI target and MALDI-time of flight (TOF)/TOF spectra acquired using a Model 4700 Proteomic Analyzer (Applied Biosystems). For the digested peptides, the mass spectrometer was operated in reflector positive ionization mode using a m/z range of 700–4000. The MS peak list was extracted in GPS explorer software using the default parameters. A list of theoretical tryptic peptides obtained with the program GPMAW (allowing for one missed cleavage) was used to interpret the MS spectra based on an average increase in m/z of 47 Da for each selenomethionine residue.
Cloning, expression and purification of the cytoplasmic domains of His6-WalK in E. coli
The cytoplasmic region of WalK (residues 208-608 TAA), with or without the H271Y mutation, was PCR amplified from NRS384 genomic DNA with primers WalK208-F/WalKTAA-R (WalKCYT) or WalK 208F/IM8/IM9/WalKTAA-R (WalKCYTH271Y), with the latter assembled by SOE-PCR. The products a After transfer of the sequence-verified construct into BL21, an overnight culture in TB of pET19b(WalKCYT) or pET19b(WalKCYTH271Y) was diluted 1:100 in 2 L of TB. Cells were grown at 37°C and 180 rpm until OD600 ˜ 0.2 and then the growth temperature was reduced to 16°C. Protein production was induced with 0.2 mM IPTG at an OD600 ≈1, then cultures were grown overnight at 16°C. Cells were harvested by centrifugation at 8000 g for 8 min at 4°C, then stored at −80°C. Frozen cells were resuspended in native lysis buffer (50 mM Tris [7.5], 500 mM NaCI, 25 mM imidazole, 5 mM EDTA), passed through a cell disruptor (Constant Systems) at 35 kpsi, then cell debris removed by centrifugation at 39,000 g for 30 min at 4°C. The supernatant was loaded on a Ni-NTA column equilibrated with equilibration buffer (25 mM Tris [7.5], 25 mM imidazole, 500 mM NaCI). The column was washed with equilibration buffer followed by a wash step with pre-elution buffer (25 mM Tris [7.5], 90 mM imidazole, 500 mM NaCI, 5 mM EDTA). WalK protein was then eluted in the same buffer containing 250 mM imidazole and immediately desalted over a HiPrep 26/10 desalting column into desalt buffer (25 mM Tris [7.5], 150 mM NaCI, 2% glycerol).
Crystalisation of the WalK PAS domain
The purified WalK-PASFULLwas concentrated to 15 mg/ml. and then screened for optimal crystallisation conditions. The protein was crystallised from conditions using the PACT screen (Molecular Dimensions). Plates were set up using a Mosquito crystallization robot (TTP Labtech). Crystallisation conditions were then refined with the best crystals formed in PAS buffer (100 mM Tris.Cl [8.0], 20 mM ZnCI2, and 26 % (w/v) PEG 3350) at 20°C. Notably, the presence of ZnCI2 was essential for crystal formation. WalK-PASFULL crystals were mounted using a 15-μM-nylon cryo-loop (Hampton Research) and soaked in cryoprotectant solutions consisting of the crystallisation PAS buffer containing 20% PEG 400 for 10 min each and flash-frozen in liquid nitrogen and stored in liquid nitrogen for X-ray diffraction studies. An initial native X-ray data set for WalK-PASFULL was collected in a cryo-stream (100 K) using an R-AXIS IVt+ image plate detector and CuKα radiation from a Rigaku free rotating anode generator (Rigaku/MSC). A total of 360 images were collected for the full dataset at ID oscillation and 10- min exposure at a distance of 90 mm from the crystal to detector. The raw data was autoindexed, integrated and scaled using Scala in the CCP4 software package. The shorter WalK-PASTRUNC domain (residues 266-371) was crystallised using the same conditions.
Model building and refinement
The structure of the PAS domain was solved by single-wavelengthanomalous dispersion (SAD) using SeMet-labelled WalK-PASFULL. A single crystal was used to collect a highly redundant dataset at the peak wavelength for selenium at the Australian Synchrotron (Table SI). Data was processed with MOSFLM52 and scaled with SCALA53. Heavy atom sites were found and refined to find the phase, and an initial model built using the AutoSol program in PHENIX54. The resulting initial model was then subjected to multiple rounds of refinement in PHENIX and rebuilding in COOT55 using a higher-resolution dataset collected with longer exposure times. The structure of the unlabeled shorter version of the WalK-PASTRUNC domain was subsequently determined by molecular replacement using PHASER52. Phasing and refinement statistics are given in Table SI.
Molecular dynamics
A membrane–bound homology model of the WalK complex (sequence based on Uniprot entry Q2G2U4) was constructed with VMD 56 and SwissModel (https://swissmodel.expasy.org) using PDB crystal structures 4MN6 (residues 266 to 320, identity 100%), 5IS1 (residues 33 to 182, identity 100%) and 4I5S (residues 223 to 599, identity 44%) as templates 57. Missing regions 1 to 33, 183 to 222 and 600 to 608 were independently modelled using secondary structure prediction program Psipred (http://bioinf.cs.ucl.ac.uk/psipred/) as a guide as no significant similar structures were available. The missing N terminal and middle sections were modelled as trans-membrane helices, while the missing C-terminus was included as disordered (Suppl. Fig. 4). Models were fully solvated, ionised with 0.15 M NaCI, and embedded through a phosphatidylcholine (POPC) lipid bilayer. One version had Zn2+ ions bound to each of the cytoplasmic PAS domains, while the other was metal free. Initial dimensions were 127 x 127 x 260 Åcontaining 385994 and 385992 atoms respectively. Molecular simulations were performed using NAMD2.12 58 with the CHARM36 force field 59.
Simulations were run with periodic boundary conditions using the ensembles at 37°C and 1 barpressure employing Langevin dynamics. Long-range Coulomb forces were computed with the Particle Mesh Ewald method with a grid spacing of 1Å. Time steps of 2 fs were used with non-bonded interactions calculated every 2 fs and full electrostatics every 4 fs while hydrogens were constrained using the SHAKE algorithm. The cut-off distance was 12 Åwith a switching distance of 10 Åand a pair-list distance of 14Å. Pressure was controlled to 1 atm using the Nose-Hoover Langevin piston method employing a piston period of 100 fs and piston decay of 50 fs. Trajectory frames were captured every 100 ps. The zinc-free model was simulated for 160 ns while the Zn2+- bound model was simulated for 200 ns. Simulations were unconstrained apart from weak harmonic constraints holding the Zn2+ ions in the bound position in the zinc-containing model. Dihedral angles between the cytoplasmic PAS and catalytic domains were measured over the course of the simulations with VMD.
MALLS analysis for molecular mass determination
PAS domains were analyzed using multi-angle laser light scattering (MALLS) to determine their mass and level of polydispersity. Proteins were first separated by gel filtration on a Superdex 75 column, then eluted protein were passed through a miniDAWN light scattering detector and OptiLab refractometer (Wyatt Technology). Weight average molecular masses were determined from the refractive index and light scattering data using the Debye fitting method (ASTRA software package, Wyatt technology).
In vitro Zn2+-loading assays
Metal loading assays were performed on purified apo-WalK-PASTRUNC and WalK-PASTRUNC H271Y (30 μM) by mixing with 10-fold molar excess Zn2+ (300 \iM ZnS04) in a total volume of 2 ml in 20 mM MOPS [7.2], 100 mM NaCI for 60 min at 4°C. The sample was desalted on a PD10 column (GE Healthcare) into the above buffer, and then the protein concentration determined. Samples containing 10 μM total protein were prepared in 3.5% HNO3 and boiled for 15 min at 95°C. Samples were then cooled and centrifuged for 20 min at 14,000 g. The supernatant was then analysed by ICP-MS (Agilent 8900 QQQ), and the protein/metal ratio was determined.
Isothermal titration calorimetry
The interaction between WalK-PASTRUNC and WalK-PASTRUNC H271Y was analysed via ITC using a MicroCal iTC200 calorimeter (Malvern Panalytical). The cell contained 50 μM protein in ITC buffer (50 mM MES [6] and 300 mM NaCI) and the syringe contained 3 mM ZnCI2 in ITC buffer. The titration was performed at 25°C with 16 injections of 3 μl with a spacing of 2 min between injections. Titrations were repeated three times. ITC data were fitted using Origin. A single-site binding model was used to fit the data to obtain the stoichiometry (n), enthalpy {ΔH), and binding affinity (Kd).
Autophosphorylation assay
WalKCYTor WalKCYT H271Y (1 μg) were incubated at room temperature in 15 μl phosphorylation buffer (25 mM Tris, 300 mM NaCI, 1 mM TCEP, 20 mM KCI, 10 mM MgCI2, pH 8). Phosphorylation reactions were started by adding 1 μl of radiolabelled ATP mixture (2.5 μCi [y-32P]-ATP and 5 μM ATP) to the protein sample, which was then incubated for 60 min at room temperature. Reactions were stopped by adding 5 μl of 3x SDS-loading buffer, then samples were analysed on a 12% SDS-PAGE gel, followed by autoradiography. The intensity of phosphorylated protein bands was determined using Quantity One software (Bio-Rad).
Detection of WalR phosphorylation using Phos-tag SDS-PAGE and Western Blot
Overnight cultures of NRS384, WalKH271Y, Δ stpl or Δ pknB containing either the empty vector pRAB11, pRAB11 WalK FLAG or pRAB11 WalK D53A FLAG were diluted 1:100 into 100 ml of TSB containing 10 μ g/ml_ chloramphenicol and 0.4 μ M ATc. Cultures were then grown to the start of stationary phase (OD600 ˜4.0). For the chromosomally tagged WalRFLAG strains, overnight TSB cultures were diluted to OD6oo = 0.01 in 1 L of TSB and samples were taken after 110 (early log), 150 (mid log), 180 (mid log), 240 (late log) and 420 min (early stationary phase). Samples were mixed with one sample volume of ice cold ethanokacetone and harvested by centrifugation at 7,300 g for 5 min at 4°C. The cells were washed with 20 ml of milliQ water and resuspended in 500 μ L of TBS (50 mM Tris.CI [7.5], 150 mM NaCI). Cells were disrupted by bead beating three times at 5,000 rpm for 30 s (Precellys 24, Bertin Instruments) and then the lysates were centrifuged at 11,000 g for 5 min at 4°C. A total of 25 μ g of protein was loaded on an 8% SDS-PAGE gel containing 50 μ M Phos-tag acrylamide (Wako Chemicals) and 100 μ M MnCI2. The gel was run according to the manufacturer’s instructions (Wako Chemicals). To remove manganese ions after electrophoresis, the gel was washed two times for 15 min with transfer buffer (25 mM Tris [8.3], 192 mM glycine, 20% methanol) containing 1 mM EDTA and once with transfer buffer without EDTA. The separated proteins were blotted onto a PVDF membrane using the Trans-Blot® Turbo™ transfer system (Bio-Rad) according to the manufacturer’s instructions. The membrane was treated with blocking buffer (5% EasyBlocker (GeneTex) in TBS, 0.05% Tween 20) for 16 h at 4°C and then with blocking buffer containing 1:500 mouse anti-FLAG® M2-Peroxidase (HRP) monoclonal antibody (Sigma) for 1 h at room temperature. The membrane was washed three times with TBS containing 0.05% Tween 20 and bound antibody was detected using the WesternSure® PREMIUM Chemiluminescent Substrate and the C-DiGit® Blot Scanner (LI-COR Biotechnology). The ratio of phosphorylated WalR was calculated by quantification of the western blot bands using GelAnalyzer 2010a.
Data availability
All sequencing data used in this study have been deposited in the National Center for Biotechnology Information BioProject database and are accessible through the BioProject accession number PRJNA486581. Atomic coordinates and data for the cytoplasmic PAS domain of WalK have been deposited in the Protein Data Bank under accession numbers 4MN5 (WalK-PASFULL; residues 251-376) and 4MN6 (WalK-PASTRUNC; residues 266–371).
Supplementary Figures
Suppl Fig 1. Purification of recombinant WalK-PASFULL. (A) SDS-PAGE gel showing different stages in the purification of WalK-PASFULL. Lane 1: whole cell lysate; lanes 2 and 3: soluble and insoluble fractions, respectively, post-cell disruption; 4: flow-through from loading soluble fraction onto GST-agarose column; 5: wash of GST-agarose column; 6: GST-agarose beads with bound GST- WalK-PAS; 7: GST-agarose beads after thrombin cleavage; 8: WalK-PASFULL eluted after thrombin cleavage of fusion protein; 9: monomer peak resulting from SEC purification of WalK-PASFULL. (B) Chromatogram from SEC purification of WalK-PASFULL showing the presence of both monomeric proteins and large aggregates; (C) Chromatogram obtained by re-chromatographing the monomer peak from panel B.
Suppl Fig 2. Incorporation of selenomethionine in WalK-PASFULLand detection by MALDI-MS. (A)
Amino acids from 251-376 of the WalK-PASFULL construct with methionine residues highlighted in red. (B) Peptide fragmentation prediction for WalK-PASFULL for trypsin digestion and the detected 24 fragments. The highlighted corrected masses were detected using MALDI-MS. (C) MALDI spectra of tryptic fragments showing the corrected mass (highlighted in purple). The molecular weight difference in the individual fragmented peptide corresponded to the number of selenomethionines in the peptide. The mass analysis verified the 100% substitution of SeMet in WalK-PASFUL domain.
Suppl Fig 3. Structural comparison of WalK-PASFULL with WalK-PASTRUNC. (A) The Zn2+ - coordinating residues of WalK-PASFULL are shown as cyan sticks, with the atoms contributing to the interactions as spheres. The coordinating bonds are illustrated with black dashed lines. The electron density shown is the 2Fº-FC map contoured at 1.5 σ. (B) Superposition of the crystal structure of WalK-PASFULL (green) with WalK-PASTRUNC (light blue) in cartoon representation. The bound Zn2+ ions are shown as spheres and the Zn2+-coordinating residues are shown as sticks. (B) The Zn2+-binding site of WalK-PASTRUNCas described in (A).
Suppl Fig 4. Homology modelling map of pdb structures used to build the 5. aureus WalK model.
Red region corresponds to structure pdb 5IS1 (residues 33-182, identity 100%), orange to pdb structure 4MN6 (residues 266 to 371, identity 100%), and underlined to pdb structure 4I5S (residues 223 to 599, identity 44%). Blue regions did not have significant similarities to the pdb. The N-terminus (1-32) was modelled as transmembrane helix, as was the middle sequence (183-222). The C-terminus was modelled as disordered.
Acknowledgements
We acknowledge funding from the Australian National Health and Medical Research Council (Project Grant GNT1010776 and Principal Research Fellowship GNT1044414 to G.F.K.; Senior Research Fellowship GNT1136021 to B.M.C.; Project Grants GNT1049192, GNT1129589 and Senior Research Fellowship GNT1105525 to T.P.S.; and Practioner Research Fellowship GNT1105905 to B.P.H.).