The structure of the bacterial oxidoreductase enzyme DsbA in complex with a peptide reveals a basis for substrate specificity in the catalytic cycle of DsbA enzymes

Oxidative protein folding in Gram-negative bacteria results in the formation of disulfide bonds between pairs of cysteine residues. This is a multistep process in which the dithiol-disulfide oxidoreductase enzyme, DsbA, plays a central role. The structure of DsbA comprises an all helical domain of unknown function and a thioredoxin domain, where active site cysteines shuttle between an oxidized, substrate-bound, reduced form and a DsbB-bound form, where DsbB is a membrane protein that reoxidizes DsbA. Most DsbA enzymes interact with a wide variety of reduced substrates and show little specificity. However, a number of DsbA enzymes have now been identified that have narrow substrate repertoires and appear to interact specifically with a smaller number of substrates. The transient nature of the DsbA-substrate complex has hampered our understanding of the factors that govern the interaction of DsbA enzymes with their substrates. Here we report the crystal structure of a complex between Escherichia coli DsbA and a peptide with a sequence derived from a substrate. The binding site identified in the DsbA-peptide complex was distinct from that observed for DsbB in the DsbA-DsbB complex. The structure revealed details of the DsbA-peptide interaction and suggested a mechanism by which DsbA can simultaneously show broad specificity for substrates yet exhibit specificity for DsbB. This mode of binding was supported by solution nuclear magnetic resonance data as well as functional data, which demonstrated that the substrate specificity of DsbA could be modified via changes at the binding interface identified in the structure of the comple

The binding of syndapin SH3 domain to dynamin proline-rich domain involves short and long distance elements

Dynamin is a GTPase that mediates vesicle fission during synaptic vesicle endocytosis. Its long C-terminal proline-rich domain contains 13 PxxP motifs, which orchestrate its interactions with multiple proteins. The SH3 domains of syndapin and endophilin bind the PxxP motifs called Site 2 and 3 (P786−P793) at the N-terminal end of the proline-rich domain, whereas the amphiphysin SH3 binds Site 9 (P833−P836) towards the C-terminal end. In some proteins, SH3/peptide interactions also involve short distance elements, which are 5−15 amino acid extensions flanking the central PxxP motif for high affinity binding. Here we found two previously unrecognised elements in the central and the C-terminal end of the dynamin proline-rich domain that account for a significant increase in syndapin binding affinity compared to a previously reported Site 2 and Site 3 PxxP peptide alone. The first new element (G807−G811) is short distance element on the C-terminal side of Site 2 PxxP, which might contact a groove identified under the RT loop of the SH3 domain. The second element (R838−P844) is located about 50 amino acids downstream of the Site 2. These two elements provide additional specificity to the syndapin SH3 domain outside of the well-described polyproline-binding groove. Thus, the dynamin:syndapin interaction is mediated via a network of multiple contacts outside the core PxxP motif over a previously unrecognised extended region of the proline-rich domain. To our knowledge this is the first example among known SH3 interactions to involve spatially separated and extended long-range elements that combine to provide a higher affinity interaction

Structural and biochemical characterisation of the oxidoreductase NmDsbA3 from Neisseria meningitidis

DsbA is an enzyme found in the periplasm of Gram-negative bacteria that catalyses the formation of disulfide bonds in a diverse array of protein substrates, many of which are involved in bacterial pathogenesis. Whilst most bacteria possess only a single essential DsbA, Neisseria meningitidis is unusual in that it possesses three DsbAs, although the reason for this additional redundancy is unclear. Two of these N. meningitidis enzymes (NmDsbA1 and NmDsbA2) play an important role in meningococcal attachment to human epithelial cells, whilst NmDsbA3 is considered to have a narrow substrate repertoire. To begin to address the role of DsbAs in the pathogenesis of N. meningitidis, we have determined the structure of NmDsbA3 to 2.3 &Aring; resolution. Although the sequence identity between NmDsbA3 and other DsbAs is low, the NmDsbA3 structure adopted a DsbA-like fold. Consistent with this finding, we demonstrated that NmDsbA3 acts as a thiol-disulfide oxidoreductase in vitro and is reoxidised by Escherichia coli DsbB (EcDsbB). However, pronounced differences in the structures between DsbA3 and EcDsbA, which are clustered around the active site of the enzyme, suggested a structural basis for the unusual substrate specificity that is observed for NmDsbA3.<br /