A mutation to Val could be tolerated as a Val can be accommodated in this region of the protein without creating severe steric clashes with the surrounding amino acids. However, the substitution creates a small cavity that could be slightly destabilizing and could explain why only half as much of this mutant is secreted compared with the WT. Once secreted, however, Microbiology inhibitor the protein is fully active both in the fluid phase and on cell surfaces. Accordingly, we found that M120V mutant was not impaired in any functional assay. On the contrary, its activity was
slightly enhanced compared with WT FI in most assays. The residue Asn133 is located in the CD5 domain, in a short α-helix, and is solvent exposed in the 3D structure of the individual domain (Fig. 8). This Asn is not glycosylated
and its substitution would seem to be tolerated in the model. However, CHIR99021 FI expression and secretion are severely impaired. Two explanations for this could be that the region around Asn133 either forms an interface with another domain of FI, or it could be important for interacting with chaperones or related proteins during its secretion and that the substitution impairs this contact. Further work will be needed to characterize this substitution at the structural level. The residue His165 is in the CD5 domain, in a loop structure and apparently fully exposed. It is partially conserved in the sequence and it could be replaced by any polar or charged side chain (Fig. 8). Its replacement with an Arg should be tolerated and our experimental data confirm this analysis since the secretion and function of FI is not affected by this mutation. On the contrary, its activity in a solution in the presence of C4BP and FH was slightly enhanced compared with WT FI. The Ala222 residue is in a loop structure and it forms a contact with Phe209. It is located next
to Cys223-Cys238 and close to the disulfide bond that links the LDLr1 domain to a short segment located prior to the FIMAC domain (Fig. 8). In this region, we have predicted a putative Ca2+-binding site, which are often present in LDLr domains. The Ala to Gly substitution Forskolin could destabilize this region of the domain and perturb the formation of the nearby disulfide bridge and/or the structure of the putative Ca2+-binding site. Such structural alterations would be consistent with the reduced secretion of this mutant that was observed experimentally and also with the observed diminished activity towards cleavage of cell bound C3b. This mutation did, however, appear to have a negligible effect on the solution-phase activity of FI. The residue Arg299 cannot be visualized in the present 3D model as it is located in a linker peptide just before the SP domain. It is possible that an Arg to Trp mutation could be tolerated fairly well in FI, as this substitution already occurs in other species.