Flexibility of prolyl oligopeptidase: Molecular dynamics and molecular framework analysis of the potential substrate pathways

Abstract

The flexibility of prolyl oligopeptidase has been investigated using molecular dynamics (MD) and molecular framework approaches to delineate the route of the substrate to the active site. The selectivity of the enzyme is mediated by a seven-bladed beta-propeller that in the crystal structure does not indicate the possible passage for the substrate to the catalytic center. Its open topology however, could allow the blades to move apart and let the substrate into the large central cavity. Flexibility analysis of prolyl oligopeptidase structure using the FIRST (Floppy Inclusion and Rigid Substructure Topology) approach and the atomic fluctuations derived from MD simulations demonstrated the rigidity of the propeller domain, which does not permit the substrate to approach the active site through this domain. Instead, a smaller tunnel at the inter-domain region comprising the highly flexible N-terminal segment of the peptidase domain and a facing hydrophilic loop from the propeller (residues 192-205) was identified by cross-correlation analysis and essential dynamics as the only potential pathway for the substrate. The functional importance of the flexible loop has been also verified by kinetic analysis of the enzyme with a split loop. Catalytic effect of engineered disulfide bridges was rationalized by characterizing the concerted motions of the two domains.

The loops facing the active site of prolyl oligopeptidase are crucial components in substrate gating and specificity

“…Crystal structures of ligand-free porcine POP in the closed state demonstrate that ligand binding is not required for domain closure, and that at least some of the enzyme molecules reside in the resting closed state. However, it is also possible that the enzyme remains closed during catalysis, requiring only movements of loop A and/or other loops and only limited inter-domain movements to allow substrate entry, as was suggested by earlier MD simulation studies [25] and by a recent study [28]. If this is the case, the open state observed in the bacterial crystal structures may be an artifact.…”

“…This mechanism suggests cooperativity of the structural dynamics of the His loop and loop A. Recent computer simulation studies suggest a mechanism whereby substrates enter the active site through a loose surface loop structure that does not involve domain separation [25], [28]. The motion and rearrangement of the flexible loop structure involving loop A and the facing loop B are postulated to regulate substrate and inhibitor entry and binding to the active site.…”

“…Limiting the flexibility between the propeller and catalytic domains with an engineered disulfide bridge inactivated the enzyme [24]. Flexibility of the loop structure at the domain interface was suggested to be required for efficient catalysis [24,25]. This loop structure comprises a loop of the propeller domain (loop A, res.…”

A study of collective atomic fluctuations and cooperativity in the U1A–RNA complex based on molecular dynamics simulations

“…The first studies utilizing this technique involved analysis of correlated motions in cytochrome c (McCammon, 1984), ribonuclease A with two different substrates (Brünger et al, 1985), HIV-1 protease (Harte et al, 1990;Harte et al, 1992;Swaminathan et al, 1991), and BPTI (Ichiye and Karplus, 1991). Since these pioneering studies, cross-correlations and DCCMs have been used to analyze the fluctuations of many diverse systems, including proteins (Arcangeli et al, 1999;Parchment and Essex, 2000;Arcangeli et al, 2001;Rizzuti et al, 2001;Young et al, 2001;Luo and Bruice, 2002;Zoete et al, 2002), protein-nucleic acid complexes (Suenaga et al, 2000;Trylska et al, 2005), and enzymes (Bahar et al, 1997;Radkiewicz and Brooks III, 2000;Rod et al, 2003;Sulpizi et al, 2003;Agarwal, 2004;Gunasekaran and Nussinov, 2004;Schiøtt, 2004;Wong et al, 2004;Fuxreiter et al, 2005;Gorfe and Caflisch, 2005;Ma et al, 2005). A recent study analyzing both the B1 domain of protein G and ubiquitin suggests the correlated motions obtained from MD simulations agree well with measured NMR data (Lange et al, 2005).…”

An Interfacial Thermodynamics Model for Protein Stability

“…Nevertheless, the RCD is useful to understand long-time scales, where small amplitude conformational deviations in substructures within a protein are neglected, such as the compression, elongation, bending or twisting of an alpha-helix. The rapid calculations for the RCD by FIRST (requiring tiny fractions of a second) has proved to be useful in making comparative studies across protein families, and to elucidate common structural features regarding flexibility important to function Rader, et al 2004;Fuxreiter, et al 2005;Costa, et al 2006;Radestock & Gohlke 2008;Mamonova et al 2008;Rader, 2010;Heal, et al 2011;Radestock & Gohlke 2011]. It has also been shown there is a statistically significant correlation between the propagation of rigidity between two mutation sites within a protein to non-additive effects in free energy cycles describing double mutant studies [Istomin, et al 2008].…”