ConSurf 2016: an improved methodology to estimate and visualize evolutionary conservation in macromolecules

Ashkenazy, Haim; Abadi, Shiran; Martz, Eric; Chay, Ofer; Mayrose, Itay; Pupko, Tal; Ben‐Tal, Nir

doi:10.1093/nar/gkw408

Cited by 2,680 publications

(2,502 citation statements)

References 74 publications

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“…The corresponding residues that were most effectively crosslinked in E. coli (11) are shown as spheres. The structure is colored according to the sequence conservation score calculated by the ConSurf server (26). The sequence alignment was generated by randomly choosing 150 amino acid sequences from the server with sequence identity to FliG between 0.25-0.5.…”

Section: Crystal Structure Of Flif-flig Complex From H Pylorimentioning

confidence: 99%

Crystal structure of the FliF–FliG complex from Helicobacter pylori yields insight into the assembly of the motor MS–C ring in the bacterial flagellum

Xue

Lam

Zhang

et al. 2018

Journal of Biological Chemistry

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The bacterial flagellar motor is a self-assembling supramolecular nanodevice.Its spontaneous biosynthesis is initiated by the insertion of the MS ring protein FliF into the inner membrane, followed by attachment of the switch protein FliG. Assembly of this multiprotein complex is tightly regulated to avoid nonspecific aggregation, but the molecular mechanisms governing flagellar assembly are unclear. Here, we present the crystal structure of the cytoplasmic domain of FliF complexed with the Nterminal domain of FliG (FliF C -FliG N ) from the bacterium Helicobacter pylori. Within this complex, FliF C interacted with FliG N through extensive hydrophobic contacts similar to those observed in the FliF C -FliG N structure from the thermophile Thermotoga maritima, indicating conservation of the FliF C -FliG N interaction across bacterial species. Analysis of the crystal lattice revealed that the heterodimeric complex packs as a linear superhelix via stacking of the armadillo-repeat-like motifs (ARM) of FliG N . Notably, this linear helix was similar to that observed for the assembly of the FliG middle domain. We validated the in vivo relevance of the FliG N stacking by complementation studies in Escherichia coli. Furthermore, structural comparison with apo FliG from the thermophile Aquifex aeolicus indicated that FliF regulates the conformational transition of FliG and exposes the complementary ARM-like motifs of FliG N , containing conserved hydrophobic residues. FliF apparently both provides a template for FliG polymerization and spatiotemporally controls subunit interactions within FliG. Our findings reveal that a small protein fold can serve as a versatile building block to assemble into a multiprotein machinery of distinct shapes for specific functions.The bacterial flagellar motor is a self-assembled reversible rotary nanodevice. This dynamic rotary motor of about 40 nm in diameter is composed of rings of protein oligomers: the L ring (outer membrane), P ring (peptidoglycan layer), MS ring (inner membrane) and C ring (cytoplasm) (1,2). The L and P rings are believed to act as a bushing through which a central rotating rod can pass. The MS and C rings contribute to the rotor part of the flagellar motor. Torque is generated by a membrane-bound stator, which converts electrochemical potential into mechanical force that acts on the rotor. The synthesis of the flagellum begins at the rotor, which is composed of the MS and C ring protein subassemblies ( Figure 1). Specifically, the assembly of the MS ring protein FliF prompts the incorporation of the switch protein FliG and subsequently the proteins FliM and FliN to form the cytoplasmic motor switch complex. The MS ring plays an important role as it interacts both with the rod where the flagellum is anchored and with the C ring where torque Crystal structure of FliF-FliG complex from H. pylori generation and rotation switching take place.In Salmonella typhimurium and Escherichia coli, there are about 26 copies of FliF in the MS ring and 26 copies of FliG, 34 c...

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Section: Crystal Structure Of Flif-flig Complex From H Pylorimentioning

confidence: 99%

Crystal structure of the FliF–FliG complex from Helicobacter pylori yields insight into the assembly of the motor MS–C ring in the bacterial flagellum

Xue

Lam

Zhang

et al. 2018

Journal of Biological Chemistry

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“…Mapping of sequence conservation on structure α1-AT 82 was performed using the Consurf 2016 server 83 using the previously designed alignment of 212 serpins 36 . Sequence coevolution analysis was performed using the OMES χ 2 residue independence test 84 , as well as the SCA 85 and ELSC 86 perturbation-based residue covariance methods.…”

Section: Sequence Methodsmentioning

confidence: 99%

Reactive Centre Loop Dynamics and Serpin Specificity

Marijanovic

Fodor

Riley

et al. 2018

Preprint

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Serine proteinase inhibitors (serpins), typically fold to a metastable native state and undergo a major conformational change in order to inhibit target proteases. However, conformational labiality of the native serpin fold renders them susceptible to misfolding and aggregation, and underlies misfolding diseases such as α1-antitrypsin deficiency. Serpin specificity towards its protease target is dictated by its flexible and solvent exposed reactive centre loop (RCL), which forms the initial interaction with the target protease during inhibition. Previous studies have attempted to alter the specificity by mutating the RCL to that of a target serpin, but the rules governing specificity are not understood well enough yet to enable specificity to be engineered at will. In this paper, we use conserpin, a synthetic, thermostable serpin, as a model protein with which to investigate the determinants of serpin specificity by engineering its RCL.Replacing the RCL sequence with that from α1-antitrypsin fails to restore specificity against trypsin or human neutrophil elastase. Structural determination of the RCL-engineered conserpin and molecular dynamics simulations indicate that, although the RCL sequence may partially dictate specificity, local electrostatics and RCL dynamics may dictate the rate of insertion during protease inhibition, and thus whether it behaves as an inhibitor or a substrate.Engineering serpin specificity is therefore substantially more complex than solely manipulating the RCL sequence, and will require a more thorough understanding of how conformational dynamics achieves the delicate balance between stability, folding and function required by the exquisite serpin mechanism of action.

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“…http://dx.doi.org/10.1101/249128 doi: bioRxiv preprint first posted online Jan. 17, 2018; Analysis of conservation using the CONSURF server 25 reveals that the bottom of the complex is the major conserved external surface, while the region of P that adorns the outside is highly variable. The relative degree of conservation is colored dark purple for highly conserved to cyan .…”

Section: Cc-by 40 International License Peer-reviewed) Is the Authormentioning

confidence: 99%

Structure of the Core of the Type Three Secretion System Export Apparatus

Kuhlen

Abrusci

Johnson

et al. 2018

Preprint

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SummaryExport of proteins through type three secretion systems is critical for bacterial motility and virulence of many major bacterial pathogens. Three putative integral membrane proteins (FliP/FliQ/FliR) are suggested to form the core of an export gate in the inner membrane, but their structure, assembly and location within the final nanomachine remain unclear. We here present the structure of this complex at 4.2 Å by cryo-electron microscopy. None of the subunits adopt canonical integral membrane protein topologies and common helix-turn-helix structural elements allow them to form a helical assembly with 5:4:1 stoichiometry. Fitting of the structure into reconstructions of intact secretion systems localize the export gate as a core component of the periplasmic portion of the machinery, and cross-linking experiments confirm this observation. This study thereby identifies the export gate as a key element of the secretion channel and implies that it primes the helical architecture of the components assembling downstream.One Sentence SummaryThe core of the T3SS export gate forms a supra-membrane helical assembly

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ConSurf 2016: an improved methodology to estimate and visualize evolutionary conservation in macromolecules

Cited by 2,680 publications

References 74 publications

Crystal structure of the FliF–FliG complex from Helicobacter pylori yields insight into the assembly of the motor MS–C ring in the bacterial flagellum

Crystal structure of the FliF–FliG complex from Helicobacter pylori yields insight into the assembly of the motor MS–C ring in the bacterial flagellum

Reactive Centre Loop Dynamics and Serpin Specificity

Structure of the Core of the Type Three Secretion System Export Apparatus

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