Biology subjects

Douzi, B.

Publications and source records attributed to Douzi, B..

4 recordsLinked to original sources

bioRxiv2022

CbpD crystal structure adds intrigue to substrate-specificity motifs in chitin-active lytic polysaccharide monooxygenases

Pseudomonas aeruginosa secretes diverse proteins via its Type 2 Secretion System, including a 39 KDa Chitin-Binding Protein, CbpD. CbpD was recently shown to be a lytic polysaccharide monooxygenase active on chitin, and to contribute substantially to virulence. To-date no structure of this virulence factor has been reported. Its first two domains are homologous to those found in the crystal structure of Vibrio cholerae GbpA, while the third domain is homologous to the NMR structure of the Cellvibrio japonicus CjLPMO10A CBM73 domain. We report the 3.0 [A] resolution crystal structure of CbpD solved by molecular replacement, which required ab initio models of each CbpD domain generated by the artificial intelligence deep learning structure prediction algorithm RoseTTAFold. The structure of CbpD confirms previously postulated chitin-specific motifs in the AA10 domain while challenging the deterministic effects of other postulated substrate specificity motifs. Additionally, the structure of CbpD shows that post translational modifications occur on the chitin binding surface. Moreover, the structure raises interesting possibilities about how Type 2 Secretion System substrates may interact with the secretion machinery and demonstrates the utility of new artificial intelligence protein structure prediction algorithms in making challenging structural targets tractable.

microbiology↗

bioRxiv2020

Multidisciplinary Interrogation of a Crucial Protein Interface in the Type II Secretion System

The type IV filament superfamily comprises widespread membrane-associated polymers in prokaryotes. The Type II secretion system (T2SS), a significant virulence pathway in many pathogens, belongs to this superfamily. A knowledge gap in the understanding of the T2SS is the molecular role of a small pseudopilin protein. Using multiple biophysical techniques, we have deciphered how this missing component of the Xcp T2SS architecture is structurally integrated, and thereby also unlocked its function. We demonstrate that the low abundance XcpH is the adapter that bridges a trimeric initiating tip complex XcpIJK with a periplasmic filament of XcpG subunits. Our model reveals that each pseudopilin protein caps an XcpG protofilament in an overall pseudopilus compatible with the dimensions of the periplasm and the outer membrane-spanning secretin through which substrates of the T2SS pass. Unexpectedly, to fulfill its adapter function, the XcpH N-terminal helix must be unwound, a property shared with the XcpG subunits. We provide the first complete structural model of a type IV filament, a result immediately transferable to understanding of other T2SS and the type IV pili.

microbiology↗

bioRxiv2020

Structural and biochemical analysis of OrfG: the VirB8-like component of the integrative and conjugative element ICESt3 from Streptococcus thermophilus

AO_SCPLOWBSTRACTC_SCPLOWConjugative transfer is a major threat to global health since it contributes to the spread of antibiotic resistance genes and virulence factors among commensal and pathogenic bacteria. To allow their transfer, mobile genetic elements including Integrative and Conjugative Elements (ICEs) use a specialized conjugative apparatus related to Type IV secretion systems (Conj-T4SS). Therefore, Conj-T4SSs are excellent targets for strategies that aim to limit the spread of antibiotic resistance. In this study, we combined structural, biochemical and biophysical approaches to study OrfG, a protein that belongs to Conj-T4SS of ICESt3 from Streptococcus thermophilus. Structural analysis of OrfG by X-ray crystallography revealed that OrfG central domain is similar to VirB8-like proteins but displays a different quaternary structure in the crystal. To understand, at a structural level, the common and the diverse features between VirB8-like proteins from both Gram-negative and -positive bacteria, we used an in silico structural alignment method that allowed us to identify different structural classes of VirB8-like proteins. Biochemical and biophysical characterizations of purified OrfG soluble domain and its central and C-terminal subdomains indicated that they are mainly monomeric in solution but able to form an unprecedented 6-mer oligomers. Our study provides new insights into the structural and assembly mode of VirB8-like proteins, a component essential for conjugative transfer and improves our understanding on these under-examined bacterial nanomachines.

biochemistry↗

bioRxiv2020

HetL provides immunity to HetR against PatS inhibition, and promotes pattern formation in the cyanobacterium Nostoc PCC 7120

Local activation and long-range inhibition are mechanisms conserved in self-organizing systems leading to biological patterns. A number of them involve the production by the developing cell of an inhibitory morphogen, but how this cell gets immune to self-inhibition is rather unknown. Under combined nitrogen starvation, the multicellular cyanobacterium Nostoc PCC 7120 develops nitrogen-fixing heterocysts with a pattern of a heterocyst every 10-12 vegetative cells. Cell differentiation is regulated by HetR which activates the synthesis of its own inhibitory morphogen (PatS), which diffusion establishes the differentiation pattern. Here we show that HetR interacts with HetL at the same interface as PatS, and that this interaction is required to suppress inhibition and to differentiate heterocysts. hetL expression is induced under nitrogen-starvation and is activated by HetR, suggesting that HetL provides immunity to the heterocyst. This protective mechanism might be conserved in other differentiating cyanobacteria as HetL homologues are spread across the phylum.

microbiology↗