The crystal structure of Clostridium perfringens SleM,a muramidase involved in cortical hydrolysis during spore germination

Clostridium perfringens spores employ two peptidoglycan lysins to degrade the spore cortex during germination. SleC initiates cortex hydrolysis to generate cortical fragments that are degraded further by the muramidase SleM. Here, we present the crystal structure of the C. perfringens S40 SleM protein at 1.8 Å. SleM comprises an N‐terminal catalytic domain that adopts an irregular α/β‐barrel fold that is common to GH25 family lysozymes, plus a C‐terminal fibronectin type III domain. The latter is involved in forming the SleM dimer that is evident in both the crystal structure and in solution. A truncated form of SleM that lacks the FnIII domain shows reduced activity against spore sacculi indicating that this domain may have a role in facilitating the position of substrate with respect to the enzyme's active site. Proteins 2016; 84:1681–1689. © 2016 Wiley Periodicals, Inc.     

Structural and functional analysis of SleL,a peptidoglycan lysin involved in germination of Bacillus spores

A major event in the germination of Bacillus spores concerns hydrolysis of the cortical peptidoglycan that surrounds the spore protoplast, the integrity of which is essential for maintenance of dormancy. Cortex degradation is initiated in all species of Bacillus spores by the combined activity of two semi‐redundant cortex‐lytic enzymes, SleB and CwlJ. A third enzyme, SleL, which has N‐acetylglucosaminidase activity, cleaves peptidoglycan fragments generated by SleB and CwlJ. Here we present crystal structures of B. cereus and B. megaterium SleL at 1.6 angstroms and 1.7 angstroms, respectively. The structures were determined with a view to identifying the structural basis of differences in catalytic efficiency between the respective enzymes. The catalytic (α/β)₈‐barrel cores of both enzymes are highly conserved from a structural perspective, including the spatial distribution of the catalytic residues. Both enzymes are equipped with two N‐terminal peptidoglycan‐binding LysM domains, which are also structurally highly conserved. However, the topological arrangement of the respective enzymes second LysM domain is markedly different, and this may account for differences in catalytic rates by impacting upon the position of the active sites with respect to their substrates. A chimeric enzyme comprising the B. megaterium SleL catalytic domain plus B. cereus SleL LysM domains displayed enzymatic activity comparable to the native B. cereus protein, exemplifying the importance of the LysM domains to SleL function. Similarly, the reciprocal construct, comprising the B. cereus SleL catalytic domain with B. megaterium SleL LysM domains, showed reduced activity compared with native B. cereus SleL. Proteins 2015; 83:1787–1799. © 2015 Wiley Periodicals, Inc.     

The catalytic domain of the germination-specific lytic transglycosylase SleB from Bacillus anthracis displays a unique active site topology

Bacillus anthracis produces metabolically inactive spores. Germination of these spores requires germination‐specific lytic enzymes (GSLEs) that degrade the unique cortex peptidoglycan to permit resumption of metabolic activity and outgrowth. We report the first crystal structure of the catalytic domain of a GSLE, SleB. The structure revealed a transglycosylase fold with unique active site topology and permitted identification of the catalytic glutamate residue. Moreover, the structure provided insights into the molecular basis for the specificity of the enzyme for muramic‐δ‐lactam‐containing cortex peptidoglycan. The protein also contains a metal‐binding site that is positioned directly at the entrance of the substrate‐binding cleft. Proteins 2012;. © 2012 Wiley Periodicals, Inc.     

Characterization of the germination of Bacillus megaterium spores lacking enzymes that degrade the spore cortex

Aims:  To determine roles of cortex lytic enzymes (CLEs) in Bacillus megaterium spore germination.
Methods and Results:  Genes for B. megaterium CLEs CwlJ and SleB were inactivated and effects of loss of one or both on germination were assessed. Loss of CwlJ or SleB did not prevent completion of germination with agents that activate the spore's germinant receptors, but loss of CwlJ slowed the release of dipicolinic acid (DPA). Loss of both CLEs also did not prevent release of DPA and glutamate during germination with KBr. However, cwlJ sleB spores had decreased viability, and could not complete germination. Loss of CwlJ eliminated spore germination with Ca²⁺ chelated to DPA (Ca-DPA), but loss of CwlJ and SleB did not affect DPA release in dodecylamine germination.
Conclusions:  CwlJ and SleB play redundant roles in cortex degradation during B. megaterium spore germination, and CwlJ accelerates DPA release and is essential for Ca-DPA germination. The roles of these CLEs are similar in germination of B. megaterium and Bacillus subtilis spores.
Significance and Impact of the Study:  These results indicate that redundant roles of CwlJ and SleB in cortex degradation during germination are similar in spores of Bacillus species; consequently, inhibition of these enzymes will prevent germination of Bacillus spores.     

Crystal structure of the peptidoglycan recognition protein at 1.8 A resolution reveals dual strategy to combat infection through two independent functional homodimers

The mammalian peptidoglycan recognition protein-S (PGRP-S) binds to peptidoglycans (PGNs), which are essential components of the cell wall of bacteria. The protein was isolated from the samples of milk obtained from camels with mastitis and purified to homogeneity and crystallized. The crystals belong to orthorhombic space group I222 with a = 87.0 Å, b = 101.7 Å and c = 162.3 Å having four crystallographically independent molecules in the asymmetric unit. The structure has been determined using X-ray crystallographic data and refined to 1.8 Å resolution. Overall, the structures of all the four crystallographically independent molecules are identical. The folding of PGRP-S consists of a central β-sheet with five β-strands, four parallel and one antiparallel, and three α-helices. This protein fold provides two functional sites. The first of these is the PGN-binding site, located on the groove that opens on the surface in the direction opposite to the location of the N terminus. The second site is implicated to be involved in the binding of non-PGN molecules, it also includes putative N-terminal segment residues (1-31) and helix α2 in the extended binding. The structure reveals a novel arrangement of PGRP-S molecules in which two pairs of molecules associate to form two independent dimers. The first dimer is formed by two molecules with N-terminal segments at the interface in which non-PGN binding sites are buried completely, whereas the PGN-binding sites of two participating molecules are fully exposed at the opposite ends of the dimer. In the second dimer, PGN-binding sites are buried at the interface while non-PGN binding sites are fully exposed at the opposite ends of the dimer. This form of dimeric arrangement is unique and seems to be aimed at enhancing the capability of the protein against specific invading bacteria. This mode of functional dimerization enhances efficiency and specificity, and is observed for the first time in the family of PGRP molecules.     

Crystal structure of MltA from Escherichia coli reveals a unique lytic transglycosylase fold

Lytic transglycosylases are bacterial enzymes involved in the maintenance and growth of the bacterial cell-wall peptidoglycan. They cleave the beta-(1,4)-glycosidic bonds in peptidoglycan forming non-reducing 1,6-anhydromuropeptides. The crystal structure of the lytic transglycosylase MltA from Escherichia coli without a membrane anchor was solved at 2.0A resolution. The enzyme has a fold completely different from those of the other known lytic transglycosylases. It contains two domains, the largest of which has a double-psi beta-barrel fold, similar to that of endoglucanase V from Humicola insolens. The smaller domain also has a beta-barrel fold topology, which is weakly related to that of the RNA-binding domain of ribosomal proteins L25 and TL5. A large groove separates the two domains, which can accommodate a glycan strand, as shown by molecular modelling. Several conserved residues, one of which is in a position equivalent to that of the catalytic acid of the H.insolens endoglucanase, flank this putative substrate-binding groove. Mutation of this residue, Asp308, abolished all activity of the enzyme, supporting the direct participation of this residue in catalysis.     

Crystal structure of the deglycating enzyme Amadoriase I in its free form and substrate‐bound complex

Amadoriases, also known as fructosyl amine oxidases (FAOX), are enzymes that catalyze the de‐glycosylation of fructosyl amino acids. As such, they are excellent candidates for the development of enzyme‐based diagnostic and therapeutic tools against age‐ and diabetes‐induced protein glycation. However, mostly because of the lack of a complete structural characterization of the different members of the family, the molecular bases of their substrate specificity have yet to be fully understood. The high resolution crystal structures of the free and the substrate‐bound form of Amadoriase I shown herein allow for the identification of key structural features that account for the diverse substrate specificity shown by this class of enzymes. This is of particular importance in the context of the rather limited and partially incomplete structural information that has so far been available in the literature on the members of the FAOX family. Moreover, using molecular dynamics simulations, we describe the tunnel conformation and the free energy profile experienced by the ligand in going from bulk water to the catalytic cavity, showing the presence of four gating helices/loops, followed by an “L‐shaped” narrow cavity. In summary, the tridimensional architecture of Amadoriase I presented herein provides a reference structural framework for the design of novel enzymes for diabetes monitoring and protein deglycation. Proteins 2016; 84:744–758. © 2016 Wiley Periodicals, Inc.     

Crystal structure of the coiled-coil domain of Drosophila TRIM protein Brat

Drosophila brain tumor (Brat) is a translational repressor belonging to the tripartite motif (TRIM) protein superfamily. During the asymmetric division of Drosophila neuroblasts, Brat localizes at the basal cortex via direct interaction with the scaffolding protein Miranda (Mira), and segregates into the basal ganglion mother cells after cell division. It was previously reported that both the coiled-coil (CC) and NHL domains of Brat are required for the interaction with Mira, but the underlying structural basis is elusive. Here, we determine the crystal structure of Brat-CC domain (aa 376-511) at 2.5 Å, showing that Brat-CC forms an elongated antiparallel dimer through an unconventional CC structure. The dimeric assembly in Brat-CC structure is similar to its counterparts in other TRIM proteins, but Brat-CC also exhibits some distinct structural features. We also demonstrate that the CC domain could not bind Mira by its own, neither does the isolated NHL domain of Brat. Rather, Brat binds to Mira through the CC-NHL domain tandem, indicating that the function of the CC domain is to assemble Brat-NHL in dimeric form, which is necessary for Mira binding.     

Crystal structure of the nucleotide‐binding domain of mortalin,the mitochondrial Hsp70 chaperone

Mortalin, a member of the Hsp70‐family of molecular chaperones, functions in a variety of processes including mitochondrial protein import and quality control, Fe‐S cluster protein biogenesis, mitochondrial homeostasis, and regulation of p53. Mortalin is implicated in regulation of apoptosis, cell stress response, neurodegeneration, and cancer and is a target of the antitumor compound MKT‐077. Like other Hsp70‐family members, Mortalin consists of a nucleotide‐binding domain (NBD) and a substrate‐binding domain. We determined the crystal structure of the NBD of human Mortalin at 2.8 Å resolution. Although the Mortalin nucleotide‐binding pocket is highly conserved relative to other Hsp70 family members, we find that its nucleotide affinity is weaker than that of Hsc70. A Parkinson's disease‐associated mutation is located on the Mortalin‐NBD surface and may contribute to Mortalin aggregation. We present structure‐based models for how the Mortalin‐NBD may interact with the nucleotide exchange factor GrpEL1, with p53, and with MKT‐077. Our structure may contribute to the understanding of disease‐associated Mortalin mutations and to improved Mortalin‐targeting antitumor compounds.     

Crystal structure of human peptidoglycan recognition protein S (PGRP-S) at 1.70 A resolution

Peptidoglycan recognition proteins (PGRPs) are pattern recognition receptors of the innate immune system that bind peptidoglycans (PGNs) of bacterial cell walls. These molecules, which are highly conserved from insects to mammals, contribute to host defense against infections by both Gram-positive and Gram-negative bacteria. Here, we present the crystal structure of human PGRP-S at 1.70A resolution. The overall structure of PGRP-S, which participates in intracellular killing of Gram-positive bacteria, is similar to that of other PGRPs, including Drosophila PGRP-LB and PGRP-SA and human PGRP-Ialpha. However, comparison with these PGRPs reveals important differences in both the PGN-binding site and a groove formed by the PGRP-specific segment on the opposite face of the molecule. This groove, which may constitute a binding site for effector or signaling proteins, is less hydrophobic and deeper in PGRP-S than in PGRP-IalphaC, whose PGRP-specific segments vary considerably in amino acid sequence. By docking a PGN ligand into the PGN-binding cleft of PGRP-S based on the known structure of a PGRP-Ialpha-PGN complex, we identified potential PGN-binding residues in PGRP-S. Differences in PGN-contacting residues and interactions suggest that, although PGRPs may engage PGNs in a similar mode, structural differences exist that likely regulate the affinity and fine specificity of PGN recognition.     

Crystal structure of aclacinomycin-10-hydroxylase, a S-adenosyl-L-methionine-dependent methyltransferase homolog involved in anthracycline biosynthesis in Streptomyces purpurascens

Anthracyclines are aromatic polyketide antibiotics, and several of these compounds are widely used as anti-tumor drugs in chemotherapy. Aclacinomycin-10-hydroxylase (RdmB) is one of the tailoring enzymes that modify the polyketide backbone in the biosynthesis of these metabolites. RdmB, a S-adenosyl-L-methionine-dependent methyltransferase homolog, catalyses the hydroxylation of 15-demethoxy-epsilon-rhodomycin to beta-rhodomycin, one step in rhodomycin biosynthesis in Streptomyces purpurascens. The crystal structure of RdmB, determined by multiwavelength anomalous diffraction to 2.1A resolution, reveals that the enzyme subunit has a fold similar to methyltransferases and binds S-adenosyl-L-methionine. The N-terminal domain, which consists almost exclusively of alpha-helices, is involved in dimerization. The C-terminal domain contains a typical alpha/beta nucleotide-binding fold, which binds S-adenosyl-L-methionine, and several of the residues interacting with the cofactor are conserved in O-methyltransferases. Adjacent to the S-adenosyl-L-methionine molecule there is a large cleft extending to the enzyme surface of sufficient size to bind the substrate. Analysis of the putative substrate-binding pocket suggests that there is no enzymatic group in proximity of the substrate 15-demethoxy-epsilon-rhodomycin, which could assist in proton abstraction and thus facilitate methyl transfer. The lack of a suitably positioned catalytic base might thus be one of the features responsible for the inability of the enzyme to act as a methyltransferase.     

Crystal structure of the N-terminal domain of MukB: a protein involved in chromosome partitioning.

BACKGROUND: The 170 kDa protein MukB has been implicated in ATP-dependent chromosome partitioning during cell division in Escherichia coli. MukB shares its dimeric structure and domain architecture with the ubiquitous family of SMC (structural maintenance of chromosomes) proteins that facilitate similar functions. The N-terminal domain of MukB carries a putative Walker A nucleotide-binding region and the C-terminal domain has been shown to bind to DNA. Mutant phenotypes and a domain arrangement similar to motor proteins that move on microtubules led to the suggestion that MukB might be a motor protein acting on DNA. RESULTS: We have cloned, overexpressed and crystallized a 26 kDa protein consisting of 227 N-terminal residues of MukB from E. coli. The structure has been solved using multiple anomalous dispersion and has been refined to 2.2 A resolution. The N-terminal domain of MukB has a mixed alpha/beta fold with a central six-stranded antiparallel beta sheet. The putative nucleotide-binding loop, which is part of an unexpected helix-loop-helix motif, is exposed on the surface and no nucleotide-binding pocket could be detected. CONCLUSIONS: The N-terminal domain of MukB has no similarity to the kinesin family of motor proteins or to any other nucleotide-binding protein. Together with the finding of the exposed Walker A motif this observation supports a model in which the N- and C-terminal domains come together in the dimer of MukB to form the active site. Conserved residues on one side of the molecule delineate a region of the N-terminal domain that is likely to interact with the C-terminal domain.     

Ionic and non-ionic compounds in the germination of spores of bacillus megaterium texas

Summary Germination requirements of suspensions of spores of Bacillus megaterium, Texas strain, an l-alanine-inosine type, have been examined employing a decrease in optical density as the criterion of germination. In deionized water, l-alanine and inosine were devoid of germinative powers. They were effective only in conjunction with any one of a large variety of salts. Data are given for germination by the monovalent and divalent alkali metal chlorides. The potassium halides were germinative; potassium fluoride was the best. Salts of organic acids, including fatty acids and polycarboxylic acids, were germinative. The need for inosine could be bypassed by various salts, e.g., ammonium propionate or salts of dipicolinic acid. Also, l-alanine was replaceable by a variety of amino acids, provided suitable ions were present. In the presence of magnesium chloride, sodium dipicolinate could substitute for either inosine or l-alanine, but not both. Salts of n-hexylamine and n-heptylamine bypassed the need for both l-alanine and inosine. A primary role for ions in germination is proposed and a secondary, augmentative action is attributed to l-alanine and inosine.     

Crystal structure of a plant immunophilin domain involved in regulation of MDR-type ABC transporters

We present the three-dimensional structure of the N-terminal FK506-binding protein (FKBP)-like domain of the immunophilin FKBP42 from Arabidopsis thaliana. The data provide the structural background for the explanation of key functional properties reported previously.