Xosite 1 was not translated into superiority as a serpin; HV3API RCL5 exhibited a mean k2 for -thrombin inhibition of 6.6 ?107 M-1 min-1 compared to 1.4 ?108 M-1 min-1 for HAPI RCL5. The differences uncovered between the ability of the fusion proteins to bind thrombin exosite 1 and theability to form a serpin-enzyme complex with thrombin suggested that the higher affinity HV3 motif might recruit n-Phenylpiperazine-1-carboxamide -thrombin to HV3-API proteins more effectively than to HCII 1-75-API proteins, but orient them in a less favourable way for attack by the thrombin active site on the RCL. Noting that the exosite 1-binding motif in hirudin is found on the C-terminus of the protein, we switched the fusion point of HV3 54-66 from the Nterminus of API M358R to the C-terminus of the serpin. While functional, the resulting fusion protein, with or 4-Dichloro-6-methylpyridine n-(2-Hydroxypropyl)acetamide 4-Chloro-2-methylquinoline 2-Ethynylthiophene 4-Bromo-3-methoxybenzaldehyde (R)-1-N-Boc-piperazine-3-carboxylic acid methyl ester 1-(4-Fluoro-2-methylphenyl)ethan-1-one 2-Bromo-6-methyl-4-(trifluoromethyl)pyridine 7-​Hexadecanone Furo[3 without a spacer peptide, inhibited thrombin at rates 11to 14-fold less than those achieved by HV3API M358R. C-terminal extensions are known within the serpin family in spite of the proximity of this region to the RCL, most notably in alpha-2-antiplasmin [1], and a Cterminal hexahistidine extension conferred enhanced thrombin inhibitory activity on HCII in the presence of heparin [43]. In spite of these precedents, positioning the exosite 1-binding motif of hirudin in this location did not PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/8086425 lead to enhanced function of API M358R. ThisFigure 6 Schematic comparison of reactions of thrombin with natural and engineered serpins. The upper panel depicts the reaction of thrombin (yellow) with heparin-activated HCII or HCII-API fusion protein HAPI M358R (blue). (1) Thrombin (IIa, with pie-shaped active site canyon opening and labelled exosite 1 (exo) encounters heparin-activated HCII or fusion protein HAPI-M358R, both containing HCII 1-75 (1-75, blue teardrop-shaped extension) and a Reactive Centre Loop (RCL, blue triangle) on a serpin scaffold (blue large oval). Interactions between HCII 1-75 and exosite 1 and the serpin RCL with thrombin's active site guide the serpin into productive encounter complex formation (2). Aligned encounter complex formation leads to final serpin-enzyme complex formation, with translocation of thrombin; the RCL is no longer visible due to its conversion into a -strand inserted into the body of the serpin (3). The lower panel depicts the reaction of thrombin (yellow) with HV3API M358R (blue), in which the C-terminal tert-Butyl (7-bromoheptyl)carbamate triskaidecapeptide of HV3 (blue circular extension) was substituted for HCII 1-75. For this fusion protein, initial complexes form which comprise either a sub-optimally aligned complex in which exosite 1 and HV3 interact without engagement of the RCL with thrombin's PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/17591728 active site (2a), or the appropriately aligned encounter complex forms directly (2b). Suboptimally aligned complexes from (2a) can proceed to optimal alignment (2b) but misalignment is predicted to be more likely with HV3API M358R than with HAPI M358R or heparin-activated HCII. Apart from the greater size of the serpins with respect to thrombin, diagrams are not to scale.Roddick et al. BMC Biochemistry 2013, 14:31 http://www.biomedcentral.com/1471-2091/14/Page 11 ofinferiority may have arisen for steric or conformational reasons; the HV3 motif may either have been too close to the RCL or inappropriately angled to permit simultaneous productive engagement of both exosite 1 and the active site of thrombin. Variant forms of API M358R with attached HV3 triskaidecapeptides, in either location, could bind thrombin via exosite-1; indeed, we.