Insight Derived from Molecular Dynamics Simulation into Substrate-Induced Changes in Protein Motions of Proteinase K

Abstract

Because of the significant industrial, agricultural and biotechnological importance of serine protease proteinase K, it has been extensively investigated using experimental approaches such as X-ray crystallography, site-directed mutagenesis and kinetic measurement. However, detailed aspects of enzymatic mechanism such as substrate binding, release and relevant regulation remain unstudied. Molecular dynamics (MD) simulations of the proteinase K alone and in complex with the peptide substrate AAPA were performed to investigate the effect of substrate binding on the dynamics/molecular motions of proteinase K. The results indicate that during simulations the substrate-complexed proteinase K adopt a more compact and stable conformation than the substrate-free form. Further essential dynamics (ED) analysis reveals that the major internal motions are confined within a subspace of very small dimension. Upon substrate binding, the overall flexibility of the protease is reduced; and the noticeable displacements are observed not only in substrate-binding regions but also in regions opposite the substrate-binding groove/pockets. The dynamic pockets caused by the large concerted motions are proposed to be linked to the substrate recognition, binding, orientation and product release; and the significant displacements in regions opposite the binding groove/pockets are considered to play a role in modulating the dynamics of enzymesubstrate interaction. Our simulation results complement the biochemical and structural studies, highlighting the dynamic mechanism of the functional properties of proteinase K.

Comparative thermal unfolding study of psychrophilic and mesophilic subtilisin-like serine proteases by molecular dynamics simulations

“…The region of residues 120-126 links α3 following the substrate-binding segment 100-104 and β4 preceding the substrate-binding segment 132-136, and therefore it has been proposed to act as a "hinge" responsible for modulating the orientation of the substrate-binding segments (Liu, Meng, Yang, Fu, & Zhang, 2007;Liu et al, 2010;Tao, Rao, & Liu, 2010). This region, which contains a five-residue insertion (Asn-Arg-Asn-Cys-Pro) in PRK relative to VPR, displays higher flexibility in VPR at the temperatures 300, 473, and 573 K. Although the two differential residues (Lys125 vs. Gly125 and Gly126 vs. Pro126 in PRK and VPR, respectively) are commonly characterized by complicated and variable structural environments in these two proteases at different simulation temperatures, the presences of a disulfide bridge (Cys34-Cys123) and a stable salt bridge (Asp117: Arg121) in PRK, which are due to the inserted residues Cys123 and Arg121, are likely to be responsible for the higher rigidity of this region in PRK than in VPR.…”

Physicochemical bases for protein folding, dynamics, and protein-ligand binding

“…In protein folding and protein-ligand binding studies, researchers generally focus on the conformational state of the protein, and for simplicity, x 1 , x 2 ,…, x n are regarded as variables that specify only the microscopic states of the protein [34]. These include all the dihedral angles of the protein chain, the eigenvector projections derived from essential dynamics analysis of an MD trajectory [6,35,36], the number of native contacts, end-to-end distance of the peptide chain, and an order parameter that describes the similarity of the protein structure to the native or other states [37], or any degree of freedom of the protein [34].…”

Structural and Dynamic Basis of Serine Proteases from Nematophagous Fungi for Cuticle Degradation

“…Although the physiochemical properties, optimum reaction conditions, substrate specificity, catalytic mechanism, dynamics, and structure-function relationship of the representative enzyme of the peptidase S8 family, the proteinase K, have been extensively studied (Bajorath et al, 1989;Betzel et al, 2001;Betzel et al, 1988;Betzel et al, 1986;Betzel et al, 1993;Ebeling et al, 1974;Hilz et al, 1975; S. Q. Müller et al, 1994;Pahler et al, 1984;Tao et al, 2010;Wolf et al, 1991), a thorough understanding of the above characteristics for the cuticle-degrading serine proteases from nematophagous fungi is of crucial importance in uncovering and describing the nematicidal mechanism by fungi at molecular level. In this chapter we describe how the variation in amino acid sequence affect the functional properties (such as substrate specificity and catalytic efficiency) of the cuticle-degrading proteases derived from different fungi through comprehensive investigation into the three-dimensional structural models of these enzymes, which will facilitate the improvement in biological control potential of nematophagous fungi through protein engineering or site-directed mutagenesis.…”

“…Q. Liu et al, 2010S. Q. Liu et al, , 2011Tao et al, 2010). Because excessively high temperatures will lead to thermal denaturation of the protein structure, the temperature just below the transition temperature is often the optimal reaction temperature of these proteases.…”

“…Q. Liu et al, 2010S. Q. Liu et al, , 2011Perica & Chothia, 2010;Tao et al, 2010). For the substrate-binding regions, the delicate balance between the rigidity and flexibility may play important roles in maintaining structural stability and modulating substrate binding specificity and affinity.…”