Molecular Dynamics of Mesophilic-Like Mutants of a Cold-Adapted Enzyme: Insights into Distal Effects Induced by the Mutations Elena Papaleo*, Marco Pasi¤, Matteo Tiberti, Luca De Gioia Department of Biotechnology and Biosciences, University of Milano-Bicocca, Milan, Italy Abstract Networks and clusters of intramolecular interactions, as well as their ‘‘communication’’ across the three-dimensional architecture have a prominent role in determining protein stability and function. Special attention has been dedicated to their role in thermal adaptation. In the present contribution, seven previously experimentally characterized mutants of a cold-adapted a-amylase, featuring mesophilic-like behavior, have been investigated by multiple molecular dynamics simulations, essential dynamics and analyses of correlated motions and electrostatic interactions. Our data elucidate the molecular mechanisms underlying the ability of single and multiple mutations to globally modulate dynamic properties of the cold-adapted a-amylase, including both local and complex unpredictable distal effects. Our investigation also shows, in agreement with the experimental data, that the conversion of the cold-adapted enzyme in a warm-adapted variant cannot be completely achieved by the introduction of few mutations, also providing the rationale behind these effects. Moreover, pivotal residues, which are likely to mediate the effects induced by the mutations, have been identified from our analyses, as well as a group of suitable candidates for protein engineering. In fact, a subset of residues here identified (as an isoleucine, or networks of mesophilic-like salt bridges in the proximity of the catalytic site) should be considered, in experimental studies, to get a more efficient modification of the features of the cold-adapted enzyme. Citation: Papaleo E, Pasi M, Tiberti M, De Gioia L (2011) Molecular Dynamics of Mesophilic-Like Mutants of a Cold-Adapted Enzyme: Insights into Distal Effects Induced by the Mutations. PLoS ONE 6(9): e24214. doi:10.1371/journal.pone.0024214 Editor: Franca Fraternali, King9s College London, United Kingdom Received April 25, 2011; Accepted August 2, 2011; Published September 7, 2011 Copyright: 2011 Papaleo et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by CASPUR (Consorzio Interuniversitario per le Applicazio-ni di Supercalcolo per Universita` e Ricerca) Standard HPC Grant 2010 to EP. The funders had no role in study design, data analysis, decision to publish or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: elena.papaleo@unimib.it ¤ Current address: Universite´ Lyon 1, CNRS, UMR 5086, Bases Mole´culaires et Structurales des Syste`mes Infectieux, IBCP FR3302, Lyon, France Introduction A detailed comprehension of molecular mechanisms that rule the relationship between stability, flexibility, and activity in extremophilic enzymes is of crucial importance both for funda- mental and applicative research [1–3]. Enzymes isolated from psychrophilic organisms have received particular attention from the scientific community in the last 20 years, thanks to their unique properties in terms of high activity at detrimental temperatures, low thermal stability and unusual specificity, offering a wide spectrum of industrial applications [4,5]. The increasing number of primary sequences and three- dimensional (3D) structures of enzymes from extremophiles [6– 8] has provided a suitable background to disclose molecular determinants of their structural stability. It is well established that psychrophilic enzymes use different adaptation strategies [9,10], with each protein family adopting its own structural strategy [9,11]. The molecular determinants and the exact relationships between activity, stability and flexibility in cold-adapted enzymes are still a matter of debate. In fact, the intrinsic thermolability and increased low temperature activity of psychrophilic enzymes prompt for a direct link between activity and stability [12]. Otherwise, it has been suggested that thermolability may be associated to a lack of evolutionary pressure for stable enzymes in low temperature habitats [13,14]. The existence of non-canonical cold-adapted enzymes, featuring both unusual thermal stability and high catalytic efficiency at low temperatures [15,16], along with the capability to uncouple activity and stability in in vitro evolution studies [17], make the definition of activity-stability- flexibility trade-off even more difficult. Structural flexibility and rigidity are likely to cooperate, each acting on specific areas of the enzyme structure, nevertheless, they are difficult to quantify for a small and anisotropic material such as a protein molecule [9,12]. In this context, the current view on the relationships between protein dynamics and function [18–22] suggests that protein function is rooted in the free energy landscape [19] and that fluctuations at equilibrium can influence biological functions. In fact, backbone flexibility profiles diverge slowly, being conserved both in protein family and superfamily [20,23,24]. However, a recent study has shown that warm- and cold-adapted enzymes belonging to the same family present common dynamics signature related to the same fold, but also specific differences which may reflect temperature adaptation [25]. In fact, the striking correspondence between picosecond dynamics and longer scale conformational changes suggests that the physical origin of functionally important collective motions is the fast time- scale local motions [19] and that differences in the fast fluctuations PLoS ONE | www.plosone.org 1 September 2011 | Volume 6 | Issue 9 | e24214