Crystal Structure of the CTP1L Endolysin Reveals How Its Activity Is Regulated by a Secondary Translation Product

Dunne, Matthew; Leicht, Stefan; Krichel, Boris; Mertens, Haydyn D. T.; Thompson, Andrew; Krijgsveld, Jeroen; Svergun, Dmitri I.; Gómez‐Torres, Natalia; Garde, Sonia; Uetrecht, Charlotte; Narbad, Arjan; Mayer, Melinda J.; Meijers, Rob

doi:10.1074/jbc.m115.671172

Cited by 41 publications

(71 citation statements)

References 49 publications

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“…The catalytic domain of SleM comprises an irregular α/β‐barrel, in which only the first five β‐strands are flanked by α‐helices and β8, which is antiparallel with respect to the other strands, closes the barrel. The topology of the SleM catalytic domain is therefore consistent with other GH25 family lysozymes for which structures are available . The most closely related structures, identified from the Dali server, included several bacteriophage‐associated endolysins.…”

Section: Resultssupporting

confidence: 66%

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

et al. 2016

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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.

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Section: Resultssupporting

confidence: 66%

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

et al. 2016

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“…P30 and P32 showed selfinteraction and also interact with each other, findings which suggest that both proteins may be organized in an oligomeric lytic complex. The activity of this complex might require both proteins, as in the case for a Clostridium endolysin recently reported (54), which also shows partial homology with P30. On the other hand, P30, but not P32, interacts with the spike protein P29, which indicates that the P30 N-terminal region (amino acids 1 to 162) would be required for that PPI.…”

Section: Discussionmentioning

confidence: 87%

Bam35 Tectivirus Intraviral Interaction Map Unveils New Function and Localization of Phage ORFan Proteins

Berjón-Otero

Lechuga

Mehla

et al. 2017

J Virol

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The family Tectiviridae comprises a group of tailless, icosahedral, membranecontaining bacteriophages that can be divided into two groups by their hosts, either Gram-negative or Gram-positive bacteria. While the first group is composed of PRD1 and nearly identical well-characterized lytic viruses, the second one includes more variable temperate phages, like GIL16 or Bam35, whose hosts are Bacillus cereus and related Gram-positive bacteria. In the genome of Bam35, nearly half of the 32 annotated open reading frames (ORFs) have no homologs in databases (ORFans), being putative proteins of unknown function, which hinders the understanding of their biology. With the aim of increasing knowledge about the viral proteome, we carried out a comprehensive yeast two-hybrid analysis of all the putative proteins encoded by the Bam35 genome. The resulting protein interactome comprised 76 unique interactions among 24 proteins, of which 12 have an unknown function. These results suggest that the P17 protein is the minor capsid protein of Bam35 and P24 is the penton protein, with the latter finding also being supported by iterative threading protein modeling. Moreover, the inner membrane transglycosylase protein P26 could have an additional structural role. We also detected interactions involving nonstructural proteins, such as the DNA-binding protein P1 and the genome terminal protein (P4), which was confirmed by coimmunoprecipitation of recombinant proteins. Altogether, our results provide a functional view of the Bam35 viral proteome, with a focus on the composition and organization of the viral particle.IMPORTANCE Tailless viruses of the family Tectiviridae can infect commensal and pathogenic Gram-positive and Gram-negative bacteria. Moreover, they have been proposed to be at the evolutionary origin of several groups of large eukaryotic DNA viruses and self-replicating plasmids. However, due to their ancient origin and complex diversity, many tectiviral proteins are ORFans of unknown function. Comprehensive protein-protein interaction (PPI) analysis of viral proteins can eventually disclose biological mechanisms and thus provide new insights into protein function unattainable by studying proteins one by one. Here we comprehensively describe intraviral PPIs among tectivirus Bam35 proteins determined using multivector yeast twohybrid screening, and these PPIs were further supported by the results of coimmunoprecipitation assays and protein structural models. This approach allowed us to propose new functions for known proteins and hypothesize about the biological role of the localization of some viral ORFan proteins within the viral particle that will be helpful for understanding the biology of tectiviruses infecting Gram-positive bacteria.KEYWORDS tectivirus, Bam35, yeast two-hybrid, intraviral interactome, ORFan, protein-protein interactions, PPIs

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“…SEC-purified proteins were used for all samples but the SpaO/SpaO C /OrgB complex, which was affinity-purified. Samples were loaded into home-made gold-coated glass capillaries (34), which were mounted into the nano electrospray ionization (ESI) source of a QToF 2 mass spectrometer (Waters, Manchester, UK, and MS Vision, Almere, the Netherlands) adapted for high-mass experiments (35) and operated in positive ion mode. Capillary and cone voltages of 1.3 to 1.5 kV and 110 to 150 V were applied, respectively.…”

Section: Methodsmentioning

confidence: 99%

Molecular organization of soluble type III secretion system sorting platform complexes

Bernal

Börnicke

Heidemann

et al. 2019

Preprint

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40Many medically relevant Gram-negative bacteria use the type III secretion system (T3SS) to 41 translocate effector proteins into the host for their invasion and intracellular survival. A multi-42 protein complex located at the cytosolic interface of the T3SS is proposed to act as a sorting 43 platform by selecting and targeting substrates for secretion through the system. However, the 44 precise stoichiometry and 3D organization of the sorting platform components is unknown. Here 45 we reconstitute soluble complexes of the Salmonella Typhimurium sorting platform proteins 46 including the ATPase InvC, the regulator OrgB, the protein SpaO and a recently identified 47 subunit SpaOC, which we show to be essential for the solubility of SpaO. We establish domain-48 domain interactions, determine for the first time the stoichiometry of each subunit within the 49 complexes by native mass spectrometry and gain insight into their organization using small-angle 50 X-ray scattering. Importantly, we find that in solution the assembly of SpaO/SpaOC/OrgB/InvC 51 adopts an extended L-shaped conformation resembling the sorting platform pods seen in in situ 52 cryo-electron tomography, proposing that this complex is the core building block that can be 53 conceivably assembled into higher oligomers to form the T3SS sorting platform. The determined 54 molecular arrangements of the soluble complexes of the sorting platform provide important 55 insights into its architecture and assembly. 56 refer to as SpaOC (16,17). This short product interacts with the full-length protein in other species 87 and thus could represent an additional structural component of the sorting platform (12,(18)(19)(20). 88 However, the function of SpaOC in type III secretion is elusive, and how it interacts with the 89 other subunits of the sorting platform is unknown. Moreover, the precise protein composition and 90 spatial molecular organization of the sorting platform, as well its assembly process and 91 mechanism of action in substrate sorting remain uncertain. 92In this study, we reconstitute and analyze for the first time the soluble assembling units of the 93 Salmonella Typhimurium SPI-1 sorting platform using purified proteins. We observe that SpaOC, 94 the second protein product of the gene spaO, is required for fully efficient type III function and 95 for the stability of the sorting platform complexes in solution. Using native mass spectrometry 96 (MS), small-angle X-ray scattering (SAXS) and multi-angle light scattering (MALS), we 97 characterize different substructures of the sorting platform, determining their stoichiometry and 98 association into SpaO/SpaOC/OrgB/InvC complexes. These complexes adopt an extended L-99 shaped conformation in solution that mirrors a segment of the sorting platform visualized by 100 CET. Our data present the most detailed assembly of the Salmonella Typhimurium SPI-1 sorting 101 platform in solution, reporting the conformation of what we propose is the core building block to 102 assemble the sorting platform at t...

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Crystal Structure of the CTP1L Endolysin Reveals How Its Activity Is Regulated by a Secondary Translation Product

Cited by 41 publications

References 49 publications

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

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

Bam35 Tectivirus Intraviral Interaction Map Unveils New Function and Localization of Phage ORFan Proteins

Molecular organization of soluble type III secretion system sorting platform complexes

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