6u12
VHH R303 C33A/C102A in complex withthe LRR domain of InlB
Structural highlights
FunctionINLB_LISMG Mediates the entry of L.monocytogenes into normally non-phagocytic mammalian host cells (Probable) (PubMed:11081636, PubMed:9282740). Its host receptor is hepatocyte growth factor receptor (HGF receptor, a tyrosine kinase, MET) which is tyrosine-phosphorylated in response to InlB in human, green monkey, mouse and dog cell lines (PubMed:11081636, PubMed:15049825). Downstream adapter proteins GAB1 and CBL are phosphorylated in response to InlB, which also causes cell colony scattering (PubMed:11081636). InlB binding to mammalian cells is saturable and inhibited by EDTA; InlB-coated beads can be taken up by host cells (PubMed:10747014). Complement component 1 Q subcomponent-binding protein (gC1q-R, C1QBP) might act as an InlB receptor, leading to activation of PI3-kinase in green monkey cells (PubMed:10747014). Stimulation of Tyr-phosphorylation by InlB is antagonized by C1QBP, showing that potentiation of MET signaling via the GW domains is not mediated by C1QBP; the exact role of C1QBP remains to be determined (PubMed:15049825). Stimulation of Tyr-phosphorylation of MET by InlB is potentiated by the InlB GW domains and glycosaminoglycans such as heparin; exogenously added InlB, or hepatocyte growth factor (HGF) will also substitute for bacterial InlB, suggesting InlB promotes bacterial invasion by mimicking the hormone HGF (PubMed:15049825). May stimulate phosphatidylinositol 4,5-bisphosphate 3-kinase (PI3-kinase) in green monkey cells, has less effect in humans as PI3-kinase is constitutively and highly expressed in Caco cells (Probable). Binds heparin; C1QBP and heparin seem to bind to the GW domains (PubMed:12411480).[1] [2] [3] [4] [5] [6] [7] [8] Publication Abstract from PubMedA distinguishing feature of camel (Camelus dromedarius) VHH domains are noncanonical disulfide bonds between CDR1 and CDR3. The disulfide bond may provide an evolutionary advantage, as one of the cysteines in the bond is germline encoded. It has been hypothesized that this additional disulfide bond may play a role in binding affinity by reducing the entropic penalty associated with immobilization of a long CDR3 loop upon antigen binding. To examine the role of a noncanonical disulfide bond on antigen binding and the biophysical properties of a VHH domain, we have used the VHH R303, which binds the Listeria virulence factor InlB as a model. Using site directed mutagenesis, we produced a double mutant of R303 (C33A/C102A) to remove the extra disulfide bond of the VHH R303. Antigen binding was not affected by loss of the disulfide bond, however the mutant VHH displayed reduced thermal stability (Tm = 12 degrees C lower than wild-type), and a loss of the ability to fold reversibly due to heat induced aggregation. X-ray structures of the mutant alone and in complex with InlB showed no major changes in the structure. B-factor analysis of the structures suggested that the loss of the disulfide bond elicited no major change on the flexibility of the CDR loops, and revealed no evidence of loop immobilization upon antigen binding. These results suggest that the noncanonical disulfide bond found in camel VHH may have evolved to stabilize the biophysical properties of the domain, rather than playing a significant role in antigen binding. Role of a noncanonical disulfide bond in the stability, affinity, and flexibility of a VHH specific for the Listeria virulence factor InlB.,Mendoza MN, Jian M, King MT, Brooks CL Protein Sci. 2020 Jan 24. doi: 10.1002/pro.3831. PMID:31981247[9] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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