Probing the primary structural determinants of streptokinase inter-domain linkers by site-specific substitution and deletion mutagenesis.
The bacterial protein streptokinase (SK) contains three independently folded domains (alpha, beta and gamma), interconnected by two flexible linkers with noticeable sequence homology. To investigate their primary structure requirements, the linkers were swapped amongst themselves i.e. linker 1 (between alpha and beta domains) was swapped with linker 2 (between beta and gamma domains) and vice versa. The resultant construct exhibited very low activity essentially due to an enhanced proteolytic susceptibility. However, a SK mutant with two linker 1 sequences, which was proteolytically as stable as WT-rSK retained about 10% of the plasminogen activator activity of rSK When the native sequence of each linker was substituted with 9 consecutive glycine sequences, in case of the linker 1 substitution mutant substantial activity was seen to survive, whereas the linker 2 mutant lost nearly all its activity. The optimal length of linkers was then studied through deletion mutagenesis experiments, which showed that deletion beyond three residues in either of the linkers resulted in virtually complete loss of activator activity. The effect of length of the linkers was then also examined by insertion of extraneous pentapeptide sequences having a propensity for adopting either an extended conformation or a relatively rigid conformation. The insertion of poly-Pro sequences into native linker 2 sequence caused up to 10-fold reduction in activity, whereas its effect in linker 1 was relatively minor. Interestingly, most of the linker mutants could form stable 1:1 complexes with human plasminogen. Taken together, these observations suggest that (i) the functioning of the inter-domain linkers of SK requires a critical minimal length, (ii) linker 1 is relatively more tolerant to insertions and sequence alterations, and appears to function primarily as a covalent connector between the alpha and beta domains, and (iii) the native linker 2 sequence is virtually indispensable for the activity of SK probably because of structural and/or flexibility requirements in SK action during catalysis.
Yadav
Suman
S
Institute of Microbial Technology (C.S.I.R), Sector 39-A, Chandigarh-160036, India.
Sahni
Girish
G
D019943:Amino Acid Substitution; D003001:Cloning, Molecular; D006801:Humans; D006868:Hydrolysis; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D010958:Plasminogen / Q000378:metabolism; D011487:Protein Conformation; D054730:Protein Interaction Domains and Motifs; D017434:Protein Structure, Tertiary; D013300:Streptokinase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020349:Surface Plasmon Resonance
D010446:Peptide Fragments; D010958:Plasminogen; D013300:Streptokinase