are encoded by differences in the primary sequence. Moreover, as results of recent advances both in biophysical spectroscopies and molecular dynamics (MD) simulations, it is possible to extract detailed information on coupled motions and networks of communicating residues in the 3D structure and during dynamics, thanks for example to analysis of cross-correlation of atomic fluctuations [26–28], or long-range pathway of communicating residues [28–31]. The psychrophilic chloride-dependent [32] a- amylase from the Antarctic bacterium Pseudoalteromonas haloplanktis (namely AHA) is a paradigm for the study of molecular determinants of enzyme cold-adaptation. In particular, AHA was one of the first psychrophilic enzymes for which the 3D structure was solved [33] (Figure 1), revealing the presence of three distinct structural domains (namely A, B and C). It has been suggested that AHA acquires a low conformational stability and high flexibility through a reduction or weakening of inter- and intra-domain interactions, as well as it features decreased activation enthalpy and a concomitant improvement in kcat if compared to mammalian a-amylases, as pig pancreatic a-amylase (PPA) [34]. AHA site-directed mutagenesis [35–37] has been insightful in clarifying the relationships between activity and structural stability. In particular, single and multiple mutants were investigated to restore weak intra-molecular interactions, which are present in the closest mesophilic homolog, PPA. It turns out that, the reestablishment of weak interactions in AHA produces variants with both thermal stability and catalytic efficiency shifted toward ‘‘mesophilic-like’’ values (Figure S1 in Supporting Information S1). The majority of the investigated mutations feature increase in Tm (melting temperature) and a decrease in both kcat and Km [35,37,38]. The authors speculated that structural stabilization may be related to improved rigidity of the active site: reducing its flexibility, in fact, would increase the activation energy [39], leading to a reduction of kcat values, and would constrain the ground-state of the enzyme-substrate complex to a narrower distribution of conformational states, thus lowering Km. In the present study, seven of the previously characterized AHA mutants [35,37,38] featuring the most clear-cut effects on activity Figure 1. AHA mutants engineered in order to restore interactions typical of the warm-adapted homolog, PPA. Localization of all the mutations included in the simulated AHA mutants, on the 3D structure. Secondary structure elements are indicated as cartoon, white, grey and black cartoons indicate domain A, B and C, respectively. The yellow dots and sticks indicate the localization of the catalytic triad, the spheres Ca2+ and Cl2 cofactors and the mutated residues are indicated as orange sticks. doi:10.1371/journal.pone.0024214.g001 Molecular Dynamics of Psychrophilic Enzyme Mutants and thermal stability (Figure 1, Figure S1 in Supporting Information S1), were investigated by multiple MD simulations in explicit solvent (collecting more than 0.25 ms, overall). The aim of our study is the elucidation of effects induced by the mutations in atomic details with particular attention to long range effects, as well as to identify the determinants of the uncompleted conversion of AHA mutants in mesophilic-like variants. The restored weak interactions are capable of modifying the AHA dynamics in the direction of the warm-adapted enzyme, inducing a complex array of long range effects. Our results also point out a subset of critical residues for PPA and AHA dynamics and structure, not previously identified, which can be a suitable test case for AHA protein engineering in a mesophilic-like direction. Results Local effects induced by the mutations in the AHA mutant variants A detailed structural characterization of the AHA mutant variants investigated by D’Amico et al. [35,37] was not previously carried out and, advantaged by the available dynamic framework, we have initially evaluated whether the mutations have effectively restored the targeted weak interactions. A description of local effects induced by the substitutions is also provided, by monitoring the persistence of residues in the surroundings of the mutations. The interactions hypothesized by D’Amico and coworkers were successfully restored (Figure S5–S9 in Supporting Information S1), although our dynamic analysis points out more complex effects and interaction networks present in PPA, which have to be considered and which can also explain the weak overall effects induced by some mutations on the kinetic and thermodynamic properties of AHA. The networks which are not restored in the AHA mutants, due to several amino acidic substitutions with respect to PPA, interest in particular electrostatic interactions as salt bridges and aromatic clusters. N12R mutation restores the salt bridge between R12A (R20P in PPA), on b1-a1 loop and D15A (D23P) on a1 (Figure 1, Figure S2 in Supporting Information S1). In PPA R20P forms a network with both D23P and E369P (which cannot be established in AHA due to P319A) and via its aliphatic carbons interact with hydrophobic residues in b8-a8 loop (366–370P, Figure S3 in Supporting Information S1), successfully restored in AHA. N150D mutation reestablishes, in AHA, a surface salt bridge between D150A, on a3, and K190A on a4 (Figure 1, Figure S3B in Supporting Information S1). However, in PPA the corresponding D173-K213P bridge belongs to a more complex cluster of electrostatic interactions, which extends through domain B, connecting it with the proximity of the catalytic domain, and cannot be established in AHA due to several substitutions (see below). Q164I mutation reinforces a hydrophobic cluster including a3 (150–166A), b4 (170–174A), as well as a3-b4 and a4-b5 (190–200A) loops (Figure 1, Figure S3C–D in Supporting Information S1). A clear increase in Hp is observed for AHA mutants which bear Q164I mutation (AHAQI, AHA5SS and AHA5) thanks to both local (a3) and distal (a4-b5) effects, resulting in a global increase of residues with high surrounding hydrophobicity (Hp.20 kcal/mol) (Figure 2 and 3). V196F mutation, in the proximity of Q164I and K300R mutations (Figure 1, Figure S3D and S4 in Supporting Information S1), has been engineered to re-establish two aromatic interactions between F196A (b5), and residues Y82A and F198A (corresponding to Y94P and F291P in PPA), on b3 and b5 respectively (Figure S4 in Supporting Information S1). In PPA, F229P is part of a larger cluster of aromatic and hydrophobic PLoS ONE | www.plosone.org 2 September 2011 | Volume 6 | Issue 9 | e24214