Structural Basis for Dual-Inhibition Mechanism of a Non-Classical Kazal-Type Serine Protease Inhibitor from Horseshoe Crab in Complex with Subtilisin

Serine proteases play a crucial role in host-pathogen interactions. In the innate immune system of invertebrates, multi-domain protease inhibitors are important for the regulation of host-pathogen interactions and antimicrobial activities. Serine protease inhibitors, 9.3-kDa CrSPI isoforms 1 and 2, have been identified from the hepatopancreas of the horseshoe crab, Carcinoscorpius rotundicauda. The CrSPIs were biochemically active, especially CrSPI-1, which potently inhibited subtilisin (Ki = 1.43 nM). CrSPI has been grouped with the non-classical Kazal-type inhibitors due to its unusual cysteine distribution. Here we report the crystal structure of CrSPI-1 in complex with subtilisin at 2.6 Å resolution and the results of biophysical interaction studies. The CrSPI-1 molecule has two domains arranged in an extended conformation. These two domains act as heads that independently interact with two separate subtilisin molecules, resulting in the inhibition of subtilisin activity at a ratio of 1:2 (inhibitor to protease). Each subtilisin molecule interacts with the reactive site loop from each domain of CrSPI-1 through a standard canonical binding mode and forms a single ternary complex. In addition, we propose the substrate preferences of each domain of CrSPI-1. Domain 2 is specific towards the bacterial protease subtilisin, while domain 1 is likely to interact with the host protease, Furin. Elucidation of the structure of the CrSPI-1: subtilisin (1∶2) ternary complex increases our understanding of host-pathogen interactions in the innate immune system at the molecular level and provides new strategies for immunomodulation

The search for the ideal biocatalyst

While the use of enzymes as biocatalysts to assist in the industrial manufacture of fine chemicals and pharmaceuticals has enormous potential, application is frequently limited by evolution-led catalyst traits. The advent of designer biocatalysts, produced by informed selection and mutation through recombinant DNA technology, enables production of process-compatible enzymes. However, to fully realize the potential of designer enzymes in industrial applications, it will be necessary to tailor catalyst properties so that they are optimal not only for a given reaction but also in the context of the industrial process in which the enzyme is applied

A comparison of different methods for modelling electromechanical multibody systems

Procedures for modelling multibody systems are well known and many formulations and tools are available for these types of systems. For several years, emphasis has been placed on the modelling of electromechanical systems, particularly multibody systems, such as robots, which are driven by electrical actuators. In this paper, three different unified modelling strategies, based on the virtual work principle, linear graph and bond graph theories, are presented and compared. Three examples, including non-academic applications, illustrate this comparison

Influence of the contact model on the dynamic response of the human knee joint

The goal of this work is to study the influence of the contact force model, contact geometry and contact material properties on the dynamic response of a human knee joint model. For this purpose a multibody knee model composed by two rigid bodies, the femur and the tibia, and four nonlinear spring elements that represent the main knee ligaments, is considered. The contact force models used were the Hertz, the Hunt-Crossley and the Lankarani-Nikravesh approaches. Results obtained from computational simulations show that Hertz law is less suitable to describe the dynamic response of the cartilage contact, because this pure elastic model does not account for the viscoelastic nature of the human articulations. Since knee can exhibit conformal and non-conformal contact scenarios, three different geometrical configurations for femur-tibia contact are considered, that is convex-convex spheres contact, convex-concave spheres contact and convex sphere-plane contact. The highest level of contact forces is obtained for the case of convex-convex spheres contact. As far as to the influence of the material contact properties is concerned, the dynamic response of a healthy and natural knee is analyzed and compared with three pathological and two artificial knee models. The obtained results demonstrate that the presence of the cartilage reduces significantly the knee contact forces.Fundação para a Ciência e a Tecnologia (FCT

Crystallographic analysis of counterion effects on subtilisin enzymatic action in acetonitrile

When enzymes are in low dielectric nonaqueous media, it would be expected that their charged groups would be more closely associated with counterions. There is evidence that these counterions may then affect enzymatic activity. Published crystal structures of proteins in organic solvents do not show increased numbers of associated counterions, and this might reflect the difficulty of distinguishing cations like Na+ from water molecules. In this paper, the placement of several Cs+ and Cl− ions in crystals of the serine protease subtilisin Carlsberg is presented. Ions are more readily identified crystallographically through their anomalous diffraction using softer X-rays. The protein conformation is very similar to that of the enzyme without CsCl in acetonitrile, both for the previously reported (1SCB) and our own newly determined model. No fewer than 11 defined sites for Cs+ cations and 8 Cl− anions are identified around the protein molecule, although most of these have partial occupancy and may represent nonspecific binding sites. Two Cs+ and two Cl− ions are close to the mouth of the active site cleft, where they may affect catalysis. In fact, cross-linked CsCl-treated subtilisin crystals transferred to acetonitrile show catalytic activity several fold higher than the reference crystals containing Na+. Presoaking with another large cation, choline, also increases the enzyme activity. The active site appears only minimally sterically perturbed by the ion presence around it, so alternative activation mechanisms can be suggested: an electrostatic redistribution and/or a larger hydration sphere that enhances the protein domain