catalytic diversity and cell wall binding repeats in the … · molecular microbiology (2018)...

Molecular Microbiology (2018) 110(6), 879–896 doi:10.1111/mmi.14134 First published online 13 November 2018

MicroReview

Catalytic diversity and cell wall binding repeats in the phage-encoded endolysins

Sebastian S. Broendum,1,2 Ashley M. Buckle1 and Sheena McGowan2*1 Biomedicine Discovery Institute, Department of Biochemistry and Molecular Biology, Monash University, Victoria, 3800, Australia.2 Biomedicine Discovery Institute, Department of Microbiology, Monash University, Victoria, 3800, Australia.

Summary

Bacteriophage-encoded endolysins can recognize and bind specific bacteria, and act to cleave the gly-cosidic and/or amide bonds in the peptidoglycan (PG) bacterial cell wall. Cleavage of the cell wall gen-erally results in the death of the bacteria. Their utility as bacteriolytic agents could be exploited for human and veterinary medicines as well as various biotech-nological applications. As interest grows in the com-mercial uses of these proteins, there has been much effort to successfully employ rational design and engineering to produce endolysins with bespoke properties. In this review, we interrogate the current structural data and identify structural features that would be of benefit to engineering the activity and specificity of phage endolysins. We show that the growing body of structural data can be used to pre-dict catalytic residues and mechanism of action from sequences of hypothetical endolysins, and probe the importance of secondary structure repeats in bacterial cell wall-binding domains.

Introduction

Bacteriophage-encoded endolysin enzymes act to lyse their host bacterial cell wall at the end of the bacteriophage lytic reproductive cycle (Young, 1992). Degradation of the peptidoglycan (PG) cell wall polymer results in osmotic shock, cell rupture and often death of the bacterial host (Fischetti, 2008). Exogenous application of purified endo-lysins to susceptible bacteria can result in their rapid and specific elimination (Loeffler et al., 2001; Nelson et al., 2001; Cheng et al., 2005; Schmelcher et al., 2012a). The specificity of endolysins for their bacterial host can vary from an entire bacterial genus to specificity at the strain level (Schmelcher et al., 2012a), which makes endolysins, or ‘enzybiotics’, a potential alternative or additive to our current antibiotics (Nelson et al., 2001).

Endolysins targeting Gram-positive bacteria have evolved a modular design, generally separating enzymatic activity and cell wall recognition into distinct domains, connected by flexible linkers. The N-terminal generally contains the enzymatically active domain(s) (EAD) that confer the catalytic activity of the endolysin, while the C-terminal cell wall-binding domain (CBD) enables the endolysin to selectively and specifically bind to the tar-get bacterial cell wall (Fenton et al., 2010; Schmelcher et al., 2012a). In contrast, endolysins targeting Gram-negative bacteria are generally small single-domain glob-ular proteins (molecular mass between 15 and 20 kDa) (Callewaert et al., 2011; Nelson et al., 2012); however, recently some ‘Gram-positive like’ domain arrangements have been observed (Briers et al., 2007; Fokine et al., 2008; Park et al., 2014; Maciejewska et al., 2017).

The rapid emergence of antibiotic resistant bacteria has stimulated considerable interest into the potential of enzybiotics as a non-antibiotic strategy for the control of pathogenic bacteria in human and veterinary medicine (Jun et al., 2014; Schuch et al., 2014; Briers et al., 2014; Czaplewski et al., 2016). In addition, endolysins are also attractive biotechnologies for primary industry (Hoopes et al., 2009; Schmelcher et al., 2015); to prevent food

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Accepted 14 September 2018. *For correspondence. E-mail [email protected]; Tel. (+613) 99029309.

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880 S. S. Broendum, A. M. Buckle and S. McGowan

© 2018 John Wiley & Sons Ltd, Molecular Microbiology, 110, 879–896

contamination by foodborne pathogens or food spoilage bacteria (see review [Schmelcher and Loessner, 2016]) and as diagnostic tools for rapid detection of bacteria from food, medical and environmental samples (Kretzer et al., 2007; Bai et al., 2016; Gómez-Torres et al., 2018). As interest grows in the commercial uses of these pro-teins, there has been much effort to successfully employ rational design/engineering to produce endolysins with bespoke properties (Gerstmans et al., 2018; São-José, 2018). As a result, the last 10 years has seen a consid-erable increase in the structural data available for this class of enzyme (25 new lysin structures have been deposited in the Protein Data Bank (PDB) since 2007). With this in mind, we sought to interrogate the current structural data and identify if there are identifiable fea-tures that would be of benefit in engineering the activity and/or specificity of phage endolysins. We searched the PDB (as available at 15 August 2018) using ‘lysin’ and ‘endolysin’ as keywords and limited our results to viral proteins (to remove the similar bacterial autolysin pro-teins). This search resulted in 338 hits that we manually curated to exclude structures of proteins which were not validated endolysins and non-modular endolysins, leaving 29 structures. In this review, we sort to combine analysis of the available structures with relevant biolog-ical data to probe the diverse enzymatic machinery of endolysins, as well as their cell wall-binding structures. Our findings show that the growing body of structural data can help identify catalytic residues and mechanism of action from sequences of hypothetical endolysins, as well as probe the importance of secondary structure repeats in bacterial CBDs.

A highly diverse group of enzymes are used to digest the PG cell wall

The bacterial cell wall is comprised of a highly cross-linked PG (or murein) polymer (Vollmer et al., 2008), renowned for its plasticity and complex higher order architecture (Vollmer and Seligman, 2010). In PG, long glycan chains, made up of alternating N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) linked by β(1-4) linkages, are cross-linked by peptide stems (Fig. 1 center). Therefore, to digest the bacterial cell wall, endolysins must be able to hydrolyze either or both glycosidic and amide (including peptide) bonds. This activity is also found in bacterial enzymes that can remodel and digest PG, which show a large degree of conservation with endo-lysins (see recent review, [Alcorlo et al., 2017]). The review by Alcorlo et al. revealed great diversity in the way PG remodeling enzymes are regulated and per-form their catalytic activities (Alcorlo et al., 2017). Our

investigation into phage-encoded endolysins produced similar results, and as such, we have focused on where structural conservation may be able to guide design or provide functional information for a previously unchar-acterized endolysin. To make the structural analysis of the phage endolysins EADs easily accessible, we have grouped our analysis of the multiple protein folds that possess enzymatic activity according to the PG bond they target (Fig. 1 and Table 1).

Glycosidases

Glycosidases, or glycoside hydrolases, catalyze the hydrolytic cleavage of the glycosidic bond (Lombard et al., 2014). The most famous of these enzymes is hen egg-white lysozyme (Blake et al., 1965) which cleaves the β(1-4) linkage between glycans (Chipman et al., 1967). As a result, endolysins that cleave this glycosidic linkage in PG are often generically called ‘lysozymes’. For exam-ple, the endolysin from the T7 bacteriophage continues to be called the ‘T7 lysozyme’, even though experimental evidence shows it to be an amidase (Inouye et al., 1973). The use of the generic name can cause confusion in the literature with regards to the actual enzymatic activity of the endolysin of interest. To minimize confusion and avoid names derived from biological role(s) rather than biochemical mechanism, we have adopted where pos-sible the convention of Alcorlo et al. (2017) and classi-fied glycosidase structure and function based on the Carbohydrate-Active enZymes database (CAZy, https://www.cazy.org).

N-acetylglucosaminidases cleave the glycan bond found on the reducing side of the GlcNAc (Fig. 1A). The only available structure of a phage endolysin N-acetylglucosaminidase domain is from the streptococ-cal C1 phage lysin, PlyC (McGowan et al., 2012). PlyC has two EADs and previous work from our laboratory showed that PlyCGyH could hydrolyze the sugar backbone in PG (McGowan et al., 2012). However, at the time we showed glycosyl hydrolase activity we did not recognize the structure as an N-acetylglucosaminidase (McGowan et al., 2012). A recent DALI search (Holm and Laakso, 2016) identified that the closest structural homologues to PlyCGyH are N-acetylglucosaminidase domains from the glycosyl hydrolase 73 (GH73) family. The closest homologs were the bacterial autolysins and structural comparisons of PlyCGyH and the LytB autolysin from Streptococcus pneumoniae show a compact, structur-ally conserved α-helical bundle consisting of six α-heli-ces (RMSD for 88 Cα atoms in the six α-helices = 4.3 Å), whereas the remaining part of the domain structure exhib-its high structural diversity (Fig. 2A). This conserved six helical structural core is present in all GH73 domains (Bai et al., 2014). In PlyCGyH, LytB, and the Staphylococcus

https://www.cazy.org

https://www.cazy.org

Structural analysis of phage endolysins 881


aureus autolysin E (AtlE) the conserved glutamic acid (E78, E564 and E138 respectively) has been shown to be essential for catalytic activity (McGowan et al., 2012; Bai et al., 2014; Mihelič et al., 2017).

Recent crystal structures of the catalytic domain from AtlE in complex with two short PG analogues showed that PG binds in a long groove that runs across the entire domain (Mihelič et al., 2017). A similar groove is present

Fig. 1. Different folds cleave different bonds in peptidoglycan (PG). (Center) Schematic representation of PG. The glycan polymer of alternating N-acetylmuramic acid (green, MurNAc) and N-acetylglucosamine (cyan, GlcNAc) are shown. The covalently linked stem peptide is composed of three to five alternating l- and d-form amino acids shown as orange circles. Amino acids labeled with X varies between different bacteria. The interpeptide bridge joining opposing stem peptides is indicated with an orange line (for comprehensive reviews on bacterial PG, see [Schleifer and Kandler, 1972; Vollmer and Seligman, 2010; Turner et al., 2014]). A representative structure of each EAD fold is shown in green cartoon representation and as a surface representation with sequence conservation scores from the consurf server (Landau et al., 2005; Ashkenazy et al., 2010; Celniker et al., 2013) mapped onto the surface, with deep purple the most conserved and teal the least conserved. Residues in pale yellow showed no conservation score due to insufficient sequence data. The PG cleavage site(s) for each class of endolysins are indicated with gray-dashed arrows. Known catalytic residues are shown in stick with orange carbon atoms. Zinc ions are shown as gray spheres. Ligands are shown in a gray stick representation. A. N-acetylglucosaminidase; PlyCGyH PDB ID: 4F88 residues 1-205. B. N-acetylmuramidases; Cpl-1 in complex with a tetrasaccharide-di-pentapeptide PG analogue PBD ID: 2IXU residues 1–188, AP3gp15 PDB ID: 5NM7 residues 77–264. C. Transglycosylase; gp144 in complex with chitotetraose PDB ID: 3BKV residues 70–260. D. N-acetylmuramoyl-l-alanine amidases; PlyL PDB ID: 1YB0 1–159, PlyPSA PDB ID: 1XOV residues 1–176. E. Endopeptidases; Ply500 PDB ID: 2VO9 residues 1–148. F. CHAP; LysK PDB ID: 4CSH residues 2–165. [Colour figure can be viewed at wileyonlinelibrary.com]

A

B

C

D

E

F

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Table 1. EAD structures included in the analysis

EndolysinPhage (bacterial host) Residues Substrate Biochemically verified? Resn (Å) PDB ID

Structure reference

Cleavage of glycosidic bonds

N-acetylglucosaminidases (CAZy GH73)PlyC Streptococcal C1

phage (Group C Streptococcus)

PlyCA 1–205 – No, based on structural comparison (this study)

3.3 4F88 McGowan et al. (2012)

N-acetylmuramidases (CAZy GH25)PlyB BcpI (B.

anthracis)1–190 – No, based on sequence

identity and structural comparison (Porter et al., 2007)

1.6 2NWO Porter et al. (2007)

Cpl-1 Pneumococcal Cp-1 (S. pneumoniae)

1–188 Tetrasaccharide-di-pentapeptide PG analogue

Yes (García et al., 1987) 2.12.3

1H092IXU

Hermoso et al. (2003), Pérez-Dorado et al. (2007)

Ply40 Listeria phage P40 (L. serovar 1/2, 4, 5, 6)

1–202 – No, based on sequence identity and structural comparison (Romero et al., 2018)

1.1 4JZ5 Romero et al. (2018)

CTP1L Clostridium phiCTP1 (C. tyrobutyricum)

1–190 – No, based on sequence identity (Mayer et al., 2010) and structural comparison (Dunne et al., 2016)

1.9 5A6S (Dunne et al., 2016)

Psm phiSM101 (C. perfringens)

1–207 α-GlcNAc No, based on sequence identity (Nariya et al., 2011) and structural comparison (Tamai et al., 2014)

1.91.4

4KRT4KRU

Tamai et al. (2014)

N-acetylmuramidasesAP3gp15 Phage AP3

(Burkholderia cenocepacia)

77–264 – Yes (Maciejewska et al., 2017)

1.7 5NM7 Maciejewska et al. (2017)

SPN1S SPN1S (S. typhimurium)

1–60 + 156–208

– No, based on structural comparison (Park et al., 2014) and this study

1.9 4OK7 Park et al. (2014)

Transglycosylases (CAZy GH23)Gp144 Phage phiKZ (P.

aeruginosa)70–260 chitotetraose Yes (Paradis-Bleau et

al., 2007)2.52.6

3BKH3BKV

Fokine et al. (2008)

Cleavage of amide bonds

Zinc amidases (Pfam Amidase_2, PF01510)PlyL prophage

LambdaBa02 (Bacillus anthracis)

1–159 – Yes (Low et al., 2005) 1.9 1YB0 Low et al. (2005)

PlyG Bacillus phage Gamma (B. anthracis)

1–165 – No, based on sequence identity (Schuch et al., 2002) and structural comparison (this study)

NMR 2L47 To be published

XlyA Bacillus subtilis prophage (B. subtilis)

5–157 – No, based on sequence identity (Longchamp et al., 1994) and structural comparison (Low et al., 2011)

2.2 3RDR Low et al. (2011)

LysGH15 Staphylococcal GH15 (S. aureus)

165–403 – No, based on structural comparison (Gu et al., 2014)

2.3 4OLS (Gu et al., 2014)

(Continued)



EndolysinPhage (bacterial host) Residues Substrate Biochemically verified? Resn (Å) PDB ID

Structure reference

Metallo-carboxypeptidases (MEROPS clan MD, family M15)PlyPSA Temperate phage

PSA (L. monocytogenes serovar 4,5,6)

1–176 – No, based on sequence identity (Zimmer et al., 2003) and structural comparison (Korndörfer et al., 2006)

1.8 1XOV Korndörfer et al. (2006)

CD27L ФCD27 (C. difficile)

1–179 – No, based on sequence identity (Mayer et al., 2008) and structural comparison (Mayer et al., 2011)

2 3QAY Mayer et al. (2011)

Ply500 Listeria phage A500 (L. monocy-togenes)

1–148 – Yesa (Loessner et al., 1995)

1.8 2VO9 Korndörfer et al. (2008)

Cysteine histidine-dependent amidohydrolase/peptidase (CHAP) (Pfam PF05257/MEROPS clan CA, family C51)PlyC Streptococcal C1


PlyCA 310–465

– Yes (Fischetti et al., 1972)


Ly7917 Streptococcus phi7917 (S. suis)

2–148 – No, based on structural comparison (this study)

1.9 5D74 To be published

LysK Staphylococcal phage K (S. aureus)

2–165 – Yesb (Becker et al., 2009)

1.8 4CSH Sanz-Gaitero et al. (2014)

LysGH15 Staphylococcus phage GH15 (S. aureus)

1–165 – No, based on structural comparison (this study)

2.7 4OLK Gu et al. (2014)

Shaded in gray = endolysins from bacteriophage targeting Gram-negative bacteria.

To be published = Structural coordinates available in PDB.al-alanoyl-d-glutamate endopeptidase.bHydrolyzes between the d-alanine of the tetra-peptide stem and the first glycine of the penta-glycine cross-bridge.

in all four GH73 domain enzymes (Fig. 2A), and molecular modeling supports similar binding modes for other bac-terial autolysins (Bai et al., 2014; Tamai et al., 2017). Our analysis here indicates that a similar binding groove, due

to both sequence and structure conservation, also exists in the endolysin domain PlyCGyH (Figs 1A). The glycan component of PG sits at the bottom of the substrate-bind-ing pocket, which is formed by the structurally conserved

Fig. 2. Structural similarity of PlyCGyH. A. PlyCGyH and the related structures of Streptococcus pneumoniae LytB (PDB ID: 4Q2W), Staphylococcus aureus autolysin E (PDB ID: 4PIA), and Clostridium perfringens autolysin (PDB ID: 5WQW). Structures are shown as a cartoon representation with the secondary structure elements in the same orientation. The structurally conserved α-helical bundle is shown in blue and remainder of protein in green. The conserved catalytic glutamic acid is shown in red as a stick representation. B. Structure of AtlE in complex with a muramyl dipeptide (NAM-Ala-d-GLU). AtlE is shown in surface representation, with the same colorscheme as in A, and the muramyl dipeptide in gray sticks. [Colour figure can be viewed at wileyonlinelibrary.com]

PlyCGyH LytBCat AtlECat AcpCat AtlECat+ MDPA B

Table 1. (Continued)




α-helical core. In contrast, the walls of the groove are not conserved and as a result the depth and composition of the groove varies between different structures. The struc-ture of the substrate-binding groove has been proposed to be important for selecting the correct bond in PG for cleav-age (Mihelič et al., 2017) and to contribute to substrate binding by interacting with the stem peptide of PG (Bai et al., 2014). Hence, the structural conservation across domains appears to mirror the conservation within PG; the conserved groove binds the invariable glycan backbone, whereas non-conserved regions of the protein appear to interact with the variable stem peptide. The interaction between these variable components provides a plausible mechanism by which substrate specificity is obtained.

The N-acetylmuramidase enzymes, also often referred to as lysozymes, cleave on the reducing side of the MurNAc sugar (Fig. 1B). Our analysis of the available structures of phage-encoded N-acetylmuramidase EADs identified five GH25 domains (Table 1). The biochemical activity of Cpl-1 has been biochemically verified to be an N-acetylmuramidase, whereas the EADs from Ply40, PlyB, CTP1L and Psm have been classified based on sequence alignments (Porter et al., 2007; Tamai et al., 2014; Dunne et al., 2016; Romero et al., 2018). Alignment and visual inspection of the respective EAD structures confirmed that all five domains adopt the expected irregu-lar β/α barrel fold consisting of an eight-stranded TIM-like β-barrel with six α-helices positioned around the central barrel (RMSD for 176 Cα atoms from five structures < 3.5 Å) (Fig. 3A, left and center). The catalytic residues are conserved and located at the center of the barrel in a cleft extending across the β-barrel (Fig. 3A, left and center). The structure of Cpl-1EAD bound to a PG analogue has been solved and by combining this structure with mod-eling, the authors suggested a binding site for a larger

PG polymer (Pérez-Dorado et al., 2007) (Fig. 3A, right). Analysis of the sequence and structures of several other members of the GH25 family suggest that this binding model may be applicable to all members of the GH25 family (Fig. 3) (Pérez-Dorado et al., 2007).

Interestingly, our mining of the PDB identified two N-acetylmuramidase EADs from bacteriophages tar-geting Gram-negative bacteria that do not belong to the GH25 family. The EAD from the AP3 endolysin, AP3gp15, has been confirmed biochemically to have N-acetylmuramidase activity (Maciejewska et al., 2017). This domain shows limited structural homology to three different families of lysozyme (GH22, hen egg-white lyso-zyme family; GH23 goose egg-white lysozyme and GH24, T4 lysozyme). Structure alignments suggest the closest structural homologue is hen egg-white lysozyme (GH22) (Maciejewska et al., 2017). Our investigation also identified that SPN1SEAD is a structural homolog of the AP3gp15EAD and therefore also a likely N-acetylmuramidase (Table 1). The SPN1SEAD consists of six α-helices where the third helix is located at the center of the domain in a vertical ori-entation, with the remaining five helices wrapped around its helical axis (Fig. 3B, [Park et al., 2014]). The closest structural homologues of SPN1SEAD are the GH19 chiti-nases; however, SPN1S does not possess chitinase activity (Park et al., 2014) and the closest non-chiti-nase structural homolog was AP3gp15 (DALI (Holm and Laakso, 2016) Z-score = 7). Based on sequence/structure conservation within the lysozyme families, the authors of the AP3gp15EAD study suggest that the enzyme pos-sesses a single catalytic residue (Maciejewska et al., 2017); however, structure-guided site-directed mutagene-sis of SPN1S identified a catalytic dyad composed of E49 and E58 (Park et al., 2014). Our sequence conservation analysis of SPN1S shows that these positions are highly

Fig. 3. Structural conservation in endolysins with N-acetylmuramidase activity. A. Left and center: Structural alignment of the five GH25 N-acetylmuramidase EAD structures. Structures are shown in cartoon representation and colored as follows: Ply40 (residues 1–202) purple, PlyB (residues 1–190) blue, Cpl-1 (residues 1–188) green, CTP1L (residues 1–190) orange, and Psm (residues 1–207) yellow. Right: Structure of Cpl-1(E94Q) (residues 1–188) in complex with a tetrasaccharide-di-pentapeptide PG analogue (PDB ID 2J8G). Cpl-1 structure is shown in a surface representation with the proposed PG-binding site indicated in blue and the catalytic residues (D10 and E94) in red. The PG analogue is shown in a gray stick representation. B. Structural alignment of the enzymatically active domains (EADs) from SPN1S and AP3gp15. The lysozyme core fold containing five helices is structurally conserved between the EAD from SPN1S (PDB ID: 4OK7 residues 1–60 and 156–208, green) and AP3gp15 (PDB ID: 5NM7 residues 77–264, blue). Catalytic residues (SPN1S: E49 and E58, AP3gp15: E101) are shown in stick with orange carbon atoms. [Colour figure can be viewed at wileyonlinelibrary.com]

A Top view Top viewSide view B




conserved, suggesting that this is indeed the location of the active site of this enzyme fold.

Transglycosylases

The hydrolase enzymes discussed above all utilize a simi-lar mechanism, via an activated water molecule, to cleave the glycan linkages. In contrast, the transglycosylase enzymes do not actually cleave PG (Höltje et al., 1975; Thunnissen et al., 1994). These enzymes act to degrade the PG through the formation of a non-reducing N-acetyl 1,6-anhydromuramic acid and N-acetylglucosamine at the two termini that initiates or promotes cell wall recy-cling (Suvorov et al., 2008). Cell wall recycling is critical to the survival of all bacteria, and lytic transglycosylase enzymes are common in both Gram-positive and -neg-ative bacteria. Several phage endolysins also possess transglycosylase activity (Taylor and Gorazdowska, 1974; Paradis-Bleau et al., 2007); however, our mining of the protein databank only identified a single structure from phage-encoded proteins. Gp144 has been con-firmed to be a transglycosylase by mass spectrometry (Paradis-Bleau et al., 2007) and the gp144EAD shows a significant structural similarity to the catalytic domains of family 1 lytic transglycosylases (Thunnissen et al., 1994; Thunnissen et al., 1995; Fokine et al., 2008). The lytic transglycosylase family 1 has four consensus sequence motifs that are involved in substrate binding (Scheurwater et al., 2008). While only three of these motifs (motifs I–III) are present in gp144, the structure of gp144 in complex with chitotetraose suggest that the substrate-binding site in gp144 and the transglycosylase 1 family is identical, and highly conserved (Fig. 1C) (Fokine et al., 2008).

Metal-dependent PG amidases and endopeptidases

In addition to cleavage of the sugar backbone, PG diges-tion can occur via cleavage of the amide bond between the MurNAc and the l-alanine moiety on the stem pep-tide (N-acetylmuramoyl-l-alanine amidase activity), or by hydrolysis of the peptide bonds in both the stem peptide and the interpeptide bridge (endopeptidase activity) (Fig. 1 and Table 1). Further, the cysteine, histidine-dependent amidohydrolase/peptidase (CHAP) domains are a special case, as this class of enzymes is classified based on their cleavage mechanism instead of the bond they cleave in PG and as such, we will discuss the CHAP domains separately.

Our investigation identified two different metal (zinc)-dependent N-acetylmuramoyl-l-alanine amidase folds. The most common amidase domain identified in our study was members of the Pfam amidase_2 family which is exemplified by the T7 lysozyme protein (Table 1). The EADs from PlyL, PlyG, XlyA and LysGH15 have the same fold, consisting of the canonical five-stranded, mostly

parallel, β-sheet, flanked by a varying number of α-helices (Fig. 4A) (Firczuk and Bochtler, 2007). The catalytic zinc ion (Zn2+) is coordinated by two histidines and a cyste-ine residue, which are highly conserved and are essen-tial for enzymatic activity (Fig. 4A). The Zn2+ is located in the center of a shallow groove that is proposed to be the PG-binding pocket (Low et al., 2011; Gu et al., 2014). The second Zn2+-dependent domain was the VanY d-Ala-d-Ala carboxypeptidase subfamily (MEROPS clan MD, fam-ily M15B, [Rawlings et al., 2018]) that we found in PlyPSA and CD27L (Table 1). While classified in the d-Ala-d-Ala carboxypeptidase subfamily, sequence alignments sug-gest that both proteins are N-acetylmuramoyl-l-alanine amidases (Zimmer et al., 2003; Mayer et al., 2008). Their fold consists of a six-stranded β-sheet surrounded by five α-helices (Fig. 4B) (Firczuk and Bochtler, 2007). The Zn2+-coordinating residues are highly conserved, but unlike the T7 lysozyme domain, two histidines and a glu-tamic acid coordinate the metal (Fig. 4B). Similarly, the Zn2+ is located in a groove suggested to be important for PG binding (Korndörfer et al., 2006; Firczuk and Bochtler, 2007; Mayer et al., 2011).

Excluding the CHAP domains, the only endolysin with endopeptidase activity for which a structure exists is Ply500. This endolysin displays l-Ala-d-Glu endo-peptidase activity, cleaving the stem peptide in Listeria monocytogenes between the first residue, l-Ala, and the second residue, d-Glu (Loessner et al., 1995) and belongs to MEROPS clan MD, family M15C (Rawlings et al., 2018). The Ply500EAD is formed by a three-stranded β-sheet which is bordered by four α-helices (Fig. 1E). The catalytically active Zn2+ is tetrahedrally coordinated by a water molecule, two histidine side chains and an aspar-tate, all of which are conserved in sequence and structure (Fig. 1E) (Firczuk and Bochtler, 2007). The differences in

Fig. 4. Structural conservation in endolysins with N-acetylmuramoyl-l-alanine amidases activity. A. Structural alignment of the four pfam Amidase_2 EAD structures. Structures are shown in cartoon representation and colored as follows: LysGH15 (residues 165–403) purple, PlyL (residues 1–160) blue, PlyG (residues 1–166) green and XlyA (residues 5–157) yellow. B. Structural alignment of the EAD from PlyPSA (green) and CD27L (blue) shown in cartoon representation. In both (A) and (B), known zinc coordinating residues are shown in stick with orange carbon atoms. Zinc ions are shown as gray spheres. [Colour figure can be viewed at wileyonlinelibrary.com]

BA




Zn2+ coordination and active site architecture could serve as good indicators for predicting the fold and function of endolysins for which no structural data exist.

The cysteine, histidine-dependent Amidohydrolase/peptidase (CHAP) domains

The CHAP domains (MEROPS clan CA, family C51) are a special case as these domains are not classified based on which bond in PG they cleave, but on their cat-alytic mechanism, relying on an invariant cysteine and histidine residue in their active site for substrate cleav-age (Bateman and Rawlings, 2003). The CHAP domains found in phage endolysins are an exquisite example of a practical enzyme scaffold that can be fine-tuned for activity and specificity. Some phage endolysin CHAP domains can cleave both the PG amidase and pepti-dase bonds (e.g. PlyGRCS [Linden et al., 2015]), while others possess only endopeptidase activity (e.g. LysK and by our classification, the closely related LysGH15). The CHAP domain fold comprises a small half β-barrel packed against a cluster of two helices, with the active site located in a cleft between these two subdomains (Fig. 1F and 5). Their active site consists of the con-served cysteine and histidine residue which are essential for catalytic activity (Bateman and Rawlings, 2003). In comparison to the CHAP domains from PlyC and Ly7919, the closely related EADs from LysK and LysGH15 have a long loop (35 residues) connecting the first and second α-helices (Fig. 5). This extension is conserved in other staphylococcal CHAP domains from both bacterial and phage-encoded proteins, and incorporates a 12-residue calcium-binding motif. The shorter loops in PlyC and Ly7919 do not contain a calcium-binding motif, nor do they have calcium bound in their available structures. Calcium has been shown to be important for the lytic

activity of some CHAP domains, including Ly7919, LysK and LysGH15 (Fenton et al., 2011; Gu et al., 2014; Sanz-Gaitero et al., 2014; Ji et al., 2015) suggesting that Ly7919 contains an alternative calcium-binding site. The addition of EDTA had no effect on the activity of PlyC (Hoopes et al., 2009), suggesting the CHAP domain from PlyC is not dependent on calcium. The CHAP domain is an excellent example of how the same fold can be evolved to cleave different bonds in PG and how the PG substrate can provide another layer of target specificity to phage endolysins.

Structural repeats are common in the CBDs

The CBD confers specificity to endolysins by recogniz-ing and binding to ligand molecules within the cell wall (Fischetti, 2005; Schmelcher et al., 2012a). Such spec-ificity can be broad, encompassing an entire bacterial genus or even multiple genera (e.g. PlyV12 [Yoong et al., 2004]), or narrow, restricting activity down to the serovar or even strain level (e.g. several Listeria endo-lysin CBDs [Schmelcher et al., 2010]). Endolysins with a broad host range often share sequence, and pre-sumably structural, similarity in their CBD, as they are thought to recognize conserved components of the cell wall (López and García, 2004; Schmelcher et al., 2012a). In contrast, highly specific endolysins likely bind target-specific ligands within the cell wall, and consequently, most endolysin ligands remain unknown (Loessner et al., 2002; Eugster et al., 2011; Schmelcher et al., 2012a).

Early reviews of the endolysins often reported that there is little sequence or structure conservation within the CBDs (Fischetti, 2005; Fischetti, 2008), but the growth of structural data available shows that this assumption is no longer valid. Out of 11 identified phage-derived endolysin CBDs, we could group the proteins broadly into four main classes or folds (Table 2 and Fig. 6). Investigation of sequence and structure in our current review and by others (López and García, 2004; Schmelcher et al., 2012a), suggests that there are indeed many endolysins that share homology to these four classes. As more structural data is gener-ated, our ability to understand the engagement of these enzymes with their target cell wall increases. One trend is rapidly emerging – the use of structural repeating units to act as a platform with which to engage the cell wall. All four classes of endolysin CBD that we define here show varying degrees of repetition either within a single polypeptide or protein oligomers. The mecha-nism by which this repetition is achieved is non-trivial for some endolysins, and how it provides improved activity remains unclear.

Fig. 5. Structural alignment of the CHAP domains. Structural alignment of the CHAP domains from: LysK (PDB ID: 4CSH, residues 2–165, gray) and Ly7917 (PDB ID: 5D74, residues 2–148, blue. Structures are shown in cartoon representation and the catalytic cysteine (LysK: C54 and Ly7917: C26) and histidine (LysK: H117 and Ly7917: H102) shown as sticks with orange carbon atoms. The calcium ion is shown as a light green sphere. [Colour figure can be viewed at wileyonlinelibrary.com]




Choline-binding modules

Pneumococcal endolysins possess a modular struc-ture of a single EAD and CBD, and utilize one or more choline-binding (ChBr) modules to bind to the cho-line-containing teichoic acid of the pneumococcal cell wall (Hermoso et al., 2003; López and García, 2004; Pérez-Dorado et al., 2007). Choline-binding modules are versatile supersecondary structures capable of playing different roles in the formation of choline-bind-ing sites, with each ChBr formed by ~20 amino acids found in multiple tandem copies (ranging from 4 to 18) (Fernández-Tornero et al., 2001). Extensive character-ization of the pneumococcal Cpl-1 endolysin showed

that the CBD contains two sub-domains, an N-terminal superhelical moiety and a C-terminal β-sheet involved in interactions with the EAD (Fig. 6A) (Hermoso et al., 2003; Pérez-Dorado et al., 2007). The superhelical moiety is formed by the first four ChBr repeats that stack to form the superhelix, and form choline-binding positions between the repeats. The choline-binding pockets are lined by conserved tryptophan residues (Fig. 6A). Consurf analysis of the Cpl-1CBD performed in our review identified 150 homologous proteins that target the streptococcus, clostridia and lactobacillus as well as other bacteria, and shows that the tryptophan residues are conserved throughout (Fig. 6A).

Table 2. CBD structures included in the analysis

EndolysinPhage (bacterial host) Residues

Conserved secondary structure repeats? Resolution (Å) PDB ID

Structure reference

Family: CW_binding_1 (PF01473) = Choline bindingCpl-1 Pneumococcal Cp-1

(S. pneumoniae)200–339 6× ChBr repeats 2.1

2.451H091OBA

Hermoso et al. (2003)

Family: SH3 = SH3b domainsLysGH15 Staphylococcus

phage GH15 (S. aureus)

400–495 – NMR 2MK5 Gu et al. (2014)

Ly7917 Streptococcus phi7917 (S. suis)

160–245 – 1.9 5D74 To be published

LysF1 Phage K1/420 (S. aureus)

391–495 – NMR 5O1Q Benešík et al. (2018)

PlyPSA Temperate phage PSA (L. monocy-togenes serovar 4,5,6)

183–314 2× SH3b-like repeats 1.8 1XOV (Korndörfer et al. (2006)


210–343 2× Sh3b repeats 1.9 4KRT Tamai et al. (2014)

Family: CW_7 (PF08230) and Family: PG_binding_1 (PF01471) = 3 helix bundleCpl-7 Pneumococcal Cp-7

(S. pneumoniae)199–341 3× 3-helix bundle 2.8

1.65I8L4CVD

Bustamante et al. (2017)

SPN1S SPN1S (S. typhimurium)

57–113 1× 3-helix bundle 1.9 4OK7 Park et al. (2014)

Ag3gp15 Phage AP3 (B. cenocepacia)

3–66 1× 3-helix bundle (2× DGhhGXXT motif)

1.72 5NM7 Maciejewska et al. (2017)

gp144 Phage phiKZ (P. aeruginosa)

1–70 1× 3-helix bundle (2× DGhhGXXT motif)

2.5 3BKH Fokine et al. (2008)

α/β structuresPlyC Streptococcal C1


PlyCB 8× PlyCB βββαβα proteins (octamer)



194–274 2× βαβββα repeats 1.9 5A6S Dunne et al. (2016)

CD27L ФCD27 (C. difficile) 180–270 2× βαββαβα repeats 2.24 4CU5 Dunne et al. (2014)

PlyG Bacillus phage Gamma (B. anthracis)

151–233 2× βαββαβ proteins (dimer)

NMR 2L48 To be published

Shaded in gray = endolysins from bacteriophage targeting Gram-negative bacteria.

To be published = structural coordinates available in PDB.



Bacterial SH3 (SH3b) domains

Our survey of the available phage-encoded endolysins structures identified five different proteins that each use SH3b domain(s) as their CBD (Table 2). SH3b domains generally form the characteristic SH3 β-barrel fold, which can contain between five and seven β-strands,

often arranged as two antiparallel β-sheets, connected

by linkers that can differ in length and composition

(Kamitori and Yoshida, 2015). The overall structures of

the endolysin SH3b domains vary (in terms of number

of residues, β-strands and linker lengths); however, each

could be readily identified as an SH3b (Fig. 6B). Two of

Fig. 6. Cell wall-binding domains of endolysins employ various structural repeats. A. Left: The choline-binding module from Cpl-1 in complex with two choline molecules (PDB ID: 1OBA). The N-terminal superhelical domain from Cpl-1 is shown in cyan whereas the C-terminal β-sheet domain is in pink. The conserved aromatic residues that interact with choline are shown in orange sticks, and choline is shown in gray sticks. Right: Structure of the CBD from Cpl-1 in surface representation with sequence conservation scores from the consurf server (Landau et al., 2005; Ashkenazy et al., 2010; Celniker et al., 2013) mapped onto the surface, with deep purple the most conserved and teal the least conserved. B. Different bacterial SH3 domains used for cell wall binding (LysGH15, PDB ID: 2MK5, residues 400–495), (Ly7917, PDB ID: 5D74, residues 160–245), (PlyPSA, PDB ID: 1XOV, residues 183–314), (Psm, PDB ID: 4KRT, residue 210–343). All structures are shown in cartoon representation and with individual SH3 domains colored in either cyan or pink. C. Different three-helical bundles used for cell wall binding (Cpl-7, PDB ID: 5I8L, residues 199–341), (gp144, PDB ID: 3BKH, residues 1–70), (SPN1S PDB ID: 4OK7, residues 57–113). All structures are shown in cartoon representation and individual 3 helical bundles coloured in either cyan, pink, or green. In gp144, the conserved cell wall binding DGhhGXXT motifs are shown in orange. D. Different α/β modules used for cell wall binding (PlyCB, PDB ID: 4f87), (CTP1L, PDB ID: 5A6S, residues 194-274), (PlyG, PDB ID: 2L48, residue 151-233). All structures are shown in cartoon representation and with individual polypeptides in either cyan or pink. [Colour figure can be viewed at wileyonlinelibrary.com]




the proteins, PlyPSA and Psm, employ tandem-encoded SH3b domains that either contain 2-fold symmetry (Psm, [Tamai et al., 2014]) or involves domain swapping between β-strands within the barrel (PlyPSA, [Korndörfer et al., 2006]) (Fig. 6B). PlyPSA has an internal duplica-tion, a structural conservation that is independent of amino acid sequence. The authors of the structure spec-ulate that it could be the result of gene duplication during evolution or a pick-up of another functionally equivalent coding sequence, followed by swapping of the respective ancestral leading β-strands (Korndörfer et al., 2006). The L. monocytogenes phage 500 endolysin (Ply500) has a similar genetic arrangement and is postulated to share the CBD arrangement, despite having completely differ-ent EADs (Korndörfer et al., 2008).

The SH3b domain in the antimicrobial glycylglycine endopeptidase, lysostaphin, can specifically recognize and bind the glycine-rich interpeptide bridge common to most staphylococcal strains (Gründling and Schneewind, 2006). This has led to the assumption that the use of an SH3b-fold within the CBD would allow for broad recog-nition of conserved ligands within a bacterial genus. As more structures and sequences were identified containing SH3b domains (PlyB, PlyPSA, Psm and other non-endo-lysin cell wall-binding proteins), this theory has continued and models exist of endolysins bound to PG whereby the interpeptide bridges are the binding sites for the CBDs (Tamai et al., 2014). However, the domain-swapped SH3b repeats in the CBD of the listeria endolysins appear to be specific and highly evolved to recognize a listeria-spe-cific surface ligand that is thought to be a carbohydrate and not a peptide (Korndörfer et al., 2006). This reduces the likelihood of being able to broadly classify the SH3b domains as peptide binding. The complexity of PlyPSA and Psm suggests that the use of SH3b is another mod-ular scaffold that can be evolved to bind specific ligands with high affinity.

Three-helix bundles

CBDs composed of a three-helix bundle is present in both Gram-positive and modular Gram-negative phage endolysins (Table 2). The Gram-positive targeting example, Cpl-7, uses three repeats of the three helix bundle CW_7 fold to bind to N-acetyl-d-glucosaminyl-(β1,4)-N-acetylmuramyl-l-alanyl-d-isoglutamine in PG (Bustamante et al., 2017) (Fig. 6C). Computational docking combined with saturation-transfer difference nuclear magnetic resonance spectroscopy suggest that N-acetyl-d-glucosaminyl-(β1,4)-N-acetylmuramyl-l-alanyl-d-isoglutamine binds in a shallow groove located between the two last helices of the bundle and the first one at the bottom (Bustamante et al., 2017). The inter-action is mediated by residues that are conserved in

the CW_7 family (Bustamante et al., 2017), suggesting other CW_7 domains may bind similar target ligands. The S. agalactiae phage endolysin, λSA2 possesses two CW_7 repeats in an unusual central position between an endopeptidase (N-terminal) and a glucosaminidase (C-terminal) domain (Pritchard et al., 2007). Considering the location of the PG binding site for the Cpl-7 CBD, at the connection between sugar strands and stem pep-tides, this centrally located CBD could align both EADs with their respective cleavage sites in the peptide moiety and the sugar strand.

The Gram-negative bacteria endolysin CBDs (SPN1SCBD, AP3gp15CBD and gp144CBD) are composed of single three-helix bundles (Fokine et al., 2008; Park et al., 2014; Maciejewska et al., 2017). Despite a rela-tively low sequence homology, the domains from gp144 and AP3gp15 are structurally conserved (Maciejewska et al., 2017), and contain repeats of the sequence motif D1GhhGXXT8, where h is any hydrophobic amino acid. This motif is commonly found in bacterial cell wall lytic enzymes and cell surface-associated proteins, and is hypothesized to be involved in PG binding (Briers et al., 2007; Fokine et al., 2008). In agreement with this, both AP3gp15 and gp144 CBDs were shown to bind to PG from a variety of Gram-negative bacteria (Briers et al., 2007). Similar observations were made for SPN1S (Park et al., 2014), despite the fact that its three-he-lix bundle differs in sequence and helical organisa-tion (Fig. 6C), and does not contain the conserved sequence motif founds in AP3gp15 and gp144 (Park et al., 2014). Hence, the three-helix bundle appears to be a commonly used fold for cell wall binding, but the target ligands in the cell wall may be diverse. One com-monality across different three-helix bundles is the use of repeats, either by whole domain duplication (Cpl-7), or repeating binding motifs within a single domain (AP3gp15 and gp144).

α/β multimers

While visually inspecting the structure of the different CBDs, we noticed a structural similarity between the CBDs from the streptococcus C1 phage lysin, PlyC, the clostridia endolysins CTP1L and CD27L and bacillus phage lysin G, PlyG (Table 2). The CBDs from CTP1L, CD27L and PlyG have low sequence identity, but adopt a similar arrangement of secondary structure elements consisting of a central parallel (CTP1L and CD27L) or antiparallel (PlyG) four-stranded β-sheet flanked by two helices (Dunne et al., 2014). This organization resembles the structure of monomeric PlyCB, which encodes the CBD of PlyC, that is composed of a central antiparallel four-stranded β-sheet with a helix flanking each side (Fig. 6D and [McGowan et al., 2012]). Interestingly, all of these



CBDs oligomerize to form their biologically functional assembly. CTP1L and CD27L were shown to contain a secondary translation site within the endolysin gene, pro-ducing a truncated CBD. This domain interacts mainly with the CBD of a full-length endolysin to produce the biologically functional assembly containing two copies of the CBD (Dunne et al., 2014; 2016). Disruption of the CBD dimer in CTP1L completely abolishes lytic activity, sug-gesting dimerization is essential for lytic activity (Dunne et al., 2014). PlyCB forms an octameric ring, which is hypothesized to be important for lytic activity (McGowan et al., 2012; Riley et al., 2015) and PlyG forms a dimer in its putative biological assembly (PDB ID 2L48). Hence, controlled oligomerization of these lysins may act as a functional switch, which may be a widespread endolysin regulatory mechanism among bacteriophages that target Gram-positive bacteria (Dunne et al., 2016).

Do we know what drives the potency and specificity of endolysins?

The increase in available structural data for phage-en-coded endolysins highlights the great diversity in struc-ture and function available in nature, but are we in a position to exploit this data to design or tailor their activ-ity? To produce a bacteriolytic agent, the first option would be to use isolated EADs to lyse target bacteria. Not all endolysins require a CBD for full exogenous lytic activity (Donovan et al., 2006; Mayer et al., 2012). The ability to function in the absence of a CBD has been suggested to be related to the overall charge of the EAD alone, as it appears that positively charged EADs can bind the bacterial cell wall independently of the CBD (Low et al., 2011). Therefore, it may be possible to use the extensive sequence and structural data to design EADs that can act alone. If the aim was to convert the EAD to a broad-spectrum antibacterial, it is likely that the glycosidases and N-acetylmuramoyl-l-alanine ami-dases would make better candidates as their substrate, the glycan polymer and the N-acetylmuramoyl-l-alanine bond, respectively, displays little variation between bac-teria (Schleifer and Kandler, 1972). Of the two enzyme classes, the N-acetylmuramoyl-l-alanine amidases may be the preferable broad-spectrum antibacterial agent as bacteria can be resistant or less susceptible to glycosi-dases due to modifications of the glycan polymer (Davis and Weiser, 2011). Conversely, EADs with endopeptidase activity often show genus specificity as the composition of the stem peptide at positions two and three as well as the interpeptide bridge varies in Gram-positive bac-teria (Schleifer and Kandler, 1972; Vollmer et al., 2008; Nelson et al., 2012). The increasing body of structural data will allow us to understand how the EADs use their

structural elements to achieve PG degradation. However, there are intricacies to the regulation and function of the EADs that we still need to uncover, including post-trans-lational modifications, substrate recognition and modifi-cation (reviewed in [Vollmer, 2008]) and protein–protein interactions (Alcorlo et al., 2017).

When an EAD does not work in isolation (Loessner et al., 2002; Porter et al., 2007; Huang et al., 2015; Becker et al., 2015), the modular design can facilitate simple domain swapping experiments. Schmelcher et al. demon-strated that switching the CBDs of two endolysins results in either a swap of their binding and lysis specificity (Schmelcher et al., 2011) or can provide the chimera with specificity to both targets (Diaz et al., 1991; Croux et al., 1993). Likewise, combining multiple EADs that target dif-ferent bonds in PG should also improve the activity of an engineered lysin (Loeffler and Fischetti, 2003; Loeffler et al., 2003; Becker et al., 2008; Schmelcher et al., 2012b), however, whether the effects are additive, synergistic or detrimental cannot yet be predicted. The PlyC enzyme, the most potent endolysin studied to date, naturally has two EADs (McGowan et al., 2012). Deletion of either of the two catalytic domains in PlyC reduced lytic activity to < 10% compared to the wild-type enzyme suggesting that the activity of the EADs are synergistic (McGowan et al., 2012). However, elimination of one of the two EADs in the LysK-like endolysins or the Twort-phage endo-lysin, has little to no effect on lytic activity (Sass and Bierbaum, 2007; Becker et al., 2015; Zhou et al., 2017; Benešík et al., 2018). Similarly, addition of EADs may not increase the lytic activity of endolysins. For example, LysK and lysostaphin both show strong lytic activity against Staphylococcus aureus; however, when their EADs were combined to produce a fusion protein cleaving three different bonds in PG, the lytic activity in vitro was not increased compared to the parent enzyme (Becker et al., 2016). Whether the impasse in lytic activity is because there is no synergy between the two catalytic domains, or due to suboptimal chimera design remains unknown. In addition to the number of EADs within an endolysin, the organization of the domains also appears to be important. For example, in the LysK and lysostaphin fusion protein, the order in which the EADs were combined had a signifi-cant effect on the lytic activity, suggesting that the context of different EADs could influence the potency of the endo-lysin (Becker et al., 2016).

The modularity of endolysins is generally discussed as a positive aspect for their potential use in biotechnology and medicine; however, the examples above show that their activity and specificity are more complex than what is encoded within the globular domains. The linker regions of these enzymes are slowly receiving attention due to their role in domain orientation and dynamics, raising the ques-tion as to whether they act as conduits for communication



between the domains. Deletion or alteration of the acidic linker in the mycobacteriophage D29 endolysin alters the activity and specificity of the endolysin (Pohane et al., 2015) and removal of the linker in Ply500 resulted in the endolysin being unable to bind to its target bacteria (Schmelcher et al., 2011). The length and nature of the linker were found to be important for activity of Ply500. The challenge for structural biology is that dynamics and communication cannot be easily implied from static crystal structures. Due to their inherently flexible nature,

far fewer full-length endolysin structures, in comparison to isolated domains, are even available for analysis. Our analysis identified only nine full-length modular endolysin proteins and five of them have been produced in the last four years (Table 3).

The structures of the available full-length proteins show that two of the nine endolysins are not assem-bled by a single polypeptide (Fig. 7 and Table 3). Both PlyC and CTP1L use multimeric arrangements of CBDs to provide their cell wall-binding ability (McGowan et

Table 3. Full-length modular endolysin structures available as at 15 August 2018 from phage-encoded genes (not bacterial)

Endolysin Phage (bacterial host) Single protein? Resolution (Å) PDB ID References

Gram-positive hostsCpl-1 Pneumococcal Cp-1

(S. pneumoniae)Yes 2.1 1H09 Hermoso et al.

(2003)PlyC Streptococcal C1


No. 1× PlyCA (EAD); 8× PlyCB (CBD)


PlyPSA Temperate phage PSA (L. monocytogenes Serovar 4, 5, 6)

Yes 1.8 1XOV Korndörfer et al. (2006)


No (1× CTP1L, 1× CTP1LCBD)

1.9 5A6S Dunne et al. (2016)


Yes 1.9 4KRT Tamai et al. (2014)

Ly7917 Phi7917 (S. suis) Yes 1.9 5D74 To be published

Gram-negative hostsSPN1S endolysin SPN1S (S.

typhimurium)Yes, modular 1.9 4OK7 Park et al. (2014)

Ag3gp15 Phage AP3 (B. cenocepacia)

Yes, modular 1.72 5MN7 Maciejewska et al. (2017)

gp144 Phage phiKZ (P. aeruginosa)

Yes, modular 2.5 3BKH Fokine et al. (2008)

TBP = to be published (structural coordinates available in PDB).

Fig. 7. Structures of two endolysin holoenzymes assembled from multiple polypeptides. The structure of the PlyC and CTP1L holoenzymes are depicted in cartoon representation. The PlyC holoenzyme consists of eight copies of PlyCB (CBD protein) and a single PlyCA (EAD protein). PlyCB monomers are colored alternately in pink and cyan. The PlyCA molecule is colored by domain as follows: N-terminal GyH domain (residues 1–205) in purple, linker 1 (residues 206–227) in red, the docking domain (residues 228–288) in yellow and the CHAP domain (residues 310–465) in green. Catalytic residues (E78, C333 and H420) are shown as sticks with carbon atoms in orange. The CTP1L holoenzyme consists of a complex between one copy of the full-length endolysin and one copy of the truncated C-terminal CBD (cyan). For the full-length CTP1L, the EAD is green and the CBD pink. [Colour figure can be viewed at wileyonlinelibrary.com]

PlyC CTP1L




al., 2012; Dunne et al., 2016). PlyC arranges a highly stable octameric ring, while CTP1L regulates its activ-ity via dimerization mediated by an alternative transla-tional start site (McGowan et al., 2012; Dunne et al., 2016). A possible hypothesis as to why multi-CBDs, or even repeating units within CBDs, are used regularly within the endolysins is that these arrangements would increase avidity toward their target bacteria. In terms of engineering endolysins; however, an increase in avid-ity does not necessarily correlate with a concomitant increase in bacteriolytic activity. Schmelcher et al. pro-duced a Ply500 variant that had a ~50-fold increase in functional affinity toward its target L. monocytogenes, but showed reduced lytic activity (Schmelcher et al., 2011). The authors suggest that the endolysins may require a certain degree of surface mobility for optimal lytic activity to allow the enzyme to move to new sub-strates (Schmelcher et al., 2011).

Conclusions

In the last 10 years, there has been a substantial increase in the available structural data for endolysins, providing insight into how the structure of endolysins facilitates their function. While the current structural data is starting to answer some of the fundamental questions on endolysin structure and function, our current understanding of the molecular details of their function are still modest. Both functional domains of the modular endolysins, the EADs and CBDs, show a wide diversity in structural folds. The EADs utilize dif-ferent folds to achieve hydrolysis of the various bonds within PG and there appear to be some preferences for the enzyme fold between the Gram-positive and Gram-negative bacteria. The conservation of the active sites in many EADs make it possible to predict the location of active sites, catalytic residues and substrate-binding pockets/grooves from sequence data of uncharacter-ized endolysins. However, to understand the mech-anistic details of cleavage and any subtle structural differences that may fine-tune the specificity of differ-ent catalytic domains, more structures of EADs in com-plex with PG analogs are necessary. Our investigation of the endolysin-specific CBDs available in the PDB clearly show that there are recognizable protein folds involved in cell wall binding, and that repeating units of tertiary structure are common. Understanding what controls the activity and specificity of endolysins will be the key for any future engineering efforts to modify these enzymes for specific purposes and fully exploit their bacteriolytic potential.

AcknowledgementsWe thank Mr. Blake T Riley for helpful discussions. The authors declare there are no conflicts of interest with this work.

Author contributionsSSB analyzed the data. SSB, AMB and SM wrote the manuscript.

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