PES and active site configurations for the reaction corresponding to Simulations 2–3 in Table 2.

<p><b>A. </b><b><i>Top left</i></b><b>.</b> PES of the reaction calculated using the C–O and H–N bonds as scanning coordinates. Two possible product states are visible on the PES corresponding respectively to hydrolyzed biapenem with unprotonated (labeled 3) or protonated N4 (labeled 5). The minimum energy path (MEP) from RS to unprotonated PS is traced by a red string. The MEP from this PS to the PS in which N4 is protonated is shown as a green string. <b><i>Top right</i></b><b>.</b> QM/MM energy values along the two MEPs. RS and ionized PS are separated by a barrier of ∼15 kcal/mol, and the reaction is strongly exergonic (−24 kcal/mol). Protonated product is ∼10 kcal/mol higher in energy than unprotonated product and a barrier of ∼20 kcal/mol separates the two states. The other three insets show the configurations of the active site corresponding to the RS, TS, and PS along the red MEP on the PES. At the RS (inset 1) Wat2 is present as a hydroxide ion. Coincident with the formation of a tetrahedral TS, a proton is shared between Wat1 and the hydroxide ion (inset 2). Concurrent with the opening of the ring (inset 3) the proton is transferred to Wat2. Thus, the reaction proceeds through the formation of a tetrahedral TS, and the rate-limiting step is the concurrent formation of the C–O bond while the C–N bond is broken. There is no proton transfer to His118, His196 or Asp120. <b>B. </b><b><i>Top left</i></b><b>.</b> PES of the reaction calculated using the C–O and H–N bonds as scanning coordinates. Two possible product states are visible on the PES corresponding respectively to hydrolyzed biapenem with unprotonated or protonated N4. The MEP from RS to unprotonated PS is traced by a red string. The MEP from this PS to the PS in which N4 is protonated is shown as a green string. <b><i>Top right</i></b><b>.</b> QM/MM energy values along the two MEPs. RS (labeled 1) and ionized PS (labeled 3) are separated by a barrier of ∼12 kcal/mol, and the reaction is only slightly exergonic (−3 kcal/mol). Protonated product (labeled 5) is isoenergetic with unprotonated product, but a barrier of ∼25 kcal/mol separates the two states. The other three insets show the configurations of the active site corresponding to the RS, ionized PS, and protonated PS. At the RS (inset labeled 1) both Wat1 and Wat2 are fully protonated. At the ionized PS a proton has been transferred from Wat1 to Wat2, and from Wat2 to Asp120 (inset labeled 3): thus Wat2 remains fully protonated. At the protonated PS (inset labeled 5) a proton has been transferred from Wat2 to biapenem N4, and from Asp120 to Wat2: thus also in this case Wat2 remains fully protonated. The most favorable reaction proceeds through the formation of a tetrahedral TS (labeled 2 on the PES), and the rate-limiting step is the concurrent formation of the C–O bond and the breaking of the C–N bond.</p

Biapenem Inactivation by B2 Metallo β-Lactamases: Energy Landscape of the Hydrolysis Reaction

<div><h3>Background</h3><p>A general mechanism has been proposed for metallo β-lactamases (MβLs), in which deprotonation of a water molecule near the Zn ion(s) results in the formation of a hydroxide ion that attacks the carbonyl oxygen of the β-lactam ring. However, because of the absence of X-ray structures that show the exact position of the antibiotic in the reactant state (RS) it has been difficult to obtain a definitive validation of this mechanism.</p> <h3>Methodology/Principal Findings</h3><p>We have employed a strategy to identify the RS, which does not rely on substrate docking and/or molecular dynamics. Starting from the X-ray structure of the enzyme:product complex (the product state, PS), a QM/MM scan was used to drive the reaction uphill from product back to reactant. Since in this process also the enzyme changes from PS to RS, we actually generate the enzyme:substrate complex from product and avoid the uncertainties associated with models of the reactant state. We used this strategy to study the reaction of biapenem hydrolysis by B2 MβL CphA. QM/MM simulations were carried out under 14 different ionization states of the active site, in order to generate potential energy surfaces (PESs) corresponding to a variety of possible reaction paths.</p> <h3>Conclusions/Significance</h3><p>The calculations support a model for biapenem hydrolysis by CphA, in which the nucleophile that attacks the β-lactam ring is not the water molecule located in proximity of the active site Zn, but a second water molecule, hydrogen bonded to the first one, which is used up in the reaction, and thus is not visible in the X-ray structure of the enzyme:product complex.</p> </div

Free energy differences and rate constants for individual steps in the hydrolysis reaction of biapenem corresponding to Simulation 3 in <b>Table 2</b>, as derived from the free energy profile in <b>Figure 5</b>.

<p>Free energy differences and rate constants for individual steps in the hydrolysis reaction of biapenem corresponding to Simulation 3 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055136#pone-0055136-t002" target="_blank"><b>Table 2</b></a>, as derived from the free energy profile in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055136#pone-0055136-g005" target="_blank"><b>Figure 5</b></a>.</p

MβLs and some of their substrates. A.

<p>Zinc binding sites of B1 (CcrA), B2 (CphA with carbonate), B3 (L1) β-lactamases. Structural models are from PDB entries 1A7T, 1X8I, 2AIO. <b>B.</b> Two typical carbapenems: Imipenem (left), Biapenem (right). An asterisk marks the carbon atom that replaces the sulfur of penicillins. <b>C.</b> Active site of CphA in complex with a bicyclic form of hydrolyzed biapenem (PDB entry 1X8I). Zn²⁺ coordination and hydrogen bonds are shown as thin yellow lines and dashed blue lines. The two bonds formed during the rearrangement are shown as thin green lines. Biapenem numbering is in red with the exception of the hydrogen transferred from O62 to C2 during the rearrangement. Red and blue arrows indicate the dynamic constraints applied during QM/MM geometry optimization to reverse the rearrangement and generate the open-ring form shown in panel D. <b>D.</b> QM/MM optimized model of the CphA active site in complex with hydrolyzed biapenem: in this state both N4 and the C6 carboxylate are protonated (atoms HN4 and HO7). A water molecule hydrogen bonded to Asp120 and loosely coordinated to Zn²⁺ (2.9 Å, dashed yellow bond) is labeled Wat2 because it is near the Zn2 site (Panel A) and to distinguish it from a second water molecule (Wat1) that might be involved in the reaction. Zn²⁺ has only five strong ligands, in agreement with spectroscopic data <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055136#pone.0055136-Sharma1" target="_blank">[22]</a>.</p

Free energy profile for the reaction corresponding to Simulation 3 in <b>Table 2</b>. Upper quadrant.

<p>Stationary and TS points are represented as red thick horizontal lines connected by dashed blue lines; numbers in blue (1–5) below each red line correspond to the same numbers on the PES of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055136#pone-0055136-g002" target="_blank"><b>Figure 2B</b></a>. Rate constants for the forward and reverse reaction at each step are defined next to each transition: values of these rate constants are reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055136#pone-0055136-t003" target="_blank"><b>Table 3</b></a>. The reaction coordinate axis is in arbitrary units and the stationary points are marked as follows: RS is biapenem; INT N4 is an intermediate conformation of the active site in which the β-lactam ring of biapenem is already open and N4 is ionized; PS N4 is a slightly changed conformation of the active site in which Wat2 becomes closer to hydrolyzed biapenem, but N4 is still ionized; PS NH4 is the open-ring form of biapenem with N4 protonated. <b>Lower quadrant.</b> Changes in the entropic contribution (-T*S) to the free energy profile shown in the upper quadrant. Both the free energy and the entropy profile are not on an absolute scale, but were shifted such that their smallest value would correspond to 0 on the energy axis.</p

PES and active site configurations for the reaction corresponding to simulation 9 in Table 2.

<p> <b>Top left.</b> PES of the reaction calculated using the C–O and C–N bonds as scanning coordinates. The MEP from RS to PS is traced by a red string. <b>Top right.</b> QM/MM energy values along the MEP: the reaction is exergonic (−10 kcal/mol), and an extended plateau (at ∼15 kcal/mol, labeled 2) consisting of 2–3 poorly differentiated TSs (or intermediates) separates RS (labeled 1) from PS (labeled 3). The other three insets show the configurations of the active site corresponding to the RS, TS, and PS along the MEP. At the RS (inset 1) Wat2 is absent, and Wat1 is hydrogen bonded to His118. Coincident with the formation of a tetrahedral TS, a proton is transferred from Wat1 to His118 (inset 2). Thus, the rate-limiting step is the formation of a tetrahedral intermediate (labeled INT on the PES) with a stretched C–N bond (1.8 Å); a barrier of only ∼5 kcal/mol separates this intermediate from the fully hydrolyzed product. The Zn ion retains a tetrahedral coordination throughout the reaction.</p

Accurate Simulation and Detection of Coevolution Signals in Multiple Sequence Alignments

<div><h3>Background</h3><p>While the conserved positions of a multiple sequence alignment (MSA) are clearly of interest, non-conserved positions can also be important because, for example, destabilizing effects at one position can be compensated by stabilizing effects at another position. Different methods have been developed to recognize the evolutionary relationship between amino acid sites, and to disentangle functional/structural dependencies from historical/phylogenetic ones.</p> <h3>Methodology/Principal Findings</h3><p>We have used two complementary approaches to test the efficacy of these methods. In the first approach, we have used a new program, MSAvolve, for the <em>in silico</em> evolution of MSAs, which records a detailed history of all covarying positions, and builds a global coevolution matrix as the accumulated sum of individual matrices for the positions forced to co-vary, the recombinant coevolution, and the stochastic coevolution. We have simulated over 1600 MSAs for 8 protein families, which reflect sequences of different sizes and proteins with widely different functions. The calculated coevolution matrices were compared with the coevolution matrices obtained for the same evolved MSAs with different coevolution detection methods. In a second approach we have evaluated the capacity of the different methods to predict close contacts in the representative X-ray structures of an additional 150 protein families using only experimental MSAs.</p> <h3>Conclusions/Significance</h3><p>Methods based on the identification of global correlations between pairs were found to be generally superior to methods based only on local correlations in their capacity to identify coevolving residues using either simulated or experimental MSAs. However, the significant variability in the performance of different methods with different proteins suggests that the simulation of MSAs that replicate the statistical properties of the experimental MSA can be a valuable tool to identify the coevolution detection method that is most effective in each case.</p> </div

Averaged results for 8 protein families with simulated MSAs under 500 sequences.

<p>Cumulative count of covariation events corresponding to the top scoring pairs in the coevolution matrices generated by different methods. <b>A.</b> MSAs simulated with MSAvolve: a dotted vertical line marks the total number of true covarying pairs controlled by the program (as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047108#pone-0047108-t001" target="_blank">Table 1</a>). <b>B.</b> MSAs simulated with SIMPROT: in these simulations a total number of 50 covarying pairs was used regardless of the sequence length. Since each curve of the two panels is not the average of independent replicas of the same experiment, traditional standard deviation (<i>std</i>) has no meaning in this case. The error bars for selected points <i>i</i> represent a weighted <i>std</i> (wσ_i) calculated as follows:.</p

Execution (CPU) time of different coevolution detection methods for the experimental MSA of PHBH (183 seq.×394 aa.).

<p>Execution (CPU) time of different coevolution detection methods for the experimental MSA of PHBH (183 seq.×394 aa.).</p

Coevolution matrices derived from a simulated MSA.

<p><b><i>totCOV</i></b>: total count of all coevolution events. Although this matrix is built independently during the simulated evolution of the MSA from a single ancestor, it can also be obtained as the sum of the mutCOV, covCOV, and recCOV matrices (see below). Residue pairs under coevolution constraint (true covarions) are indicated by red circles. <b><i>mutCOV</i></b>: count of coevolution events due to random point mutations at positions that are not set to coevolve. <b><i>recCOV</i></b>: count of coevolution events due to recombination. This count includes residues pairs that are true covarions. <b><i>covCOV</i></b>: count of true covarions. There are counts also at pairs of positions that were not set to be covarying because when two or more covarion pairs mutate and segregate also the cross-counts between pairs are added.</p