Figures and data
Structures and catalytic mechanism of β-lactam antibiotics and PDC-3 β-lactamase.
(A) Structures of representative β-lactam antibiotics. The notation R (R1/R2) represents the point of addition of functional groups. (B) Structures of commonly used cephalosporins. The R1 side chains of the antibiotics are shown in red, while the R2 side chains are marked in blue. (C) General mechanism of PDC-3 β-lactamase hydrolysis of cephalosporins. (D) The overall structure of the protein is shown in the cartoon representation (PDB ID: 4HEF). The Ω-loop and R2-loop are colored orange and red, respectively. The conserved residues in the active sites are colored green and highlighted as sticks.
Structural stability and dynamic flexibility analyses of wild-type PDC-3 β-lactamase and its variants.
(A) Pairwise Root Mean Square Deviation (RMSD) comparison of wild-type PDC-3 and its variants. The cross correlation matrix shows the RMSD values between each pair of structures. The color intensity represents the RMSD value, with lower values indicating a higher degree of structural similarity between the structures. This visualization can be used to identify patterns and trends in the structural stability of the set of structures and demonstrate the impact of substitutions on the overall conformational stability of the protein. (B) The root-mean-square fluctuation (RMSF) of wild-type PDC-3 and its variants. The Ω-loop (residues G183 to S226) is highlighted in yellow, and the R2-loop (residues L280 to Q310) is highlighted in blue. (C) Core Cα root-mean-square deviation (RMSD) superimposition from PDC-3 and its mutants. The blue parts represent the least mobile Cα atoms (80%) while the red parts highlight the most mobile atoms (20%).
E219K and Y221A mutations reshape the catalytic conformations and protonation states of K67.
(A) Density distribution of the x1 torsion angles of S64 (left) and K67 (right) in wild-type PDC-3 and its variants. (B) Hydrogen bond interactions (dashed lines) between K67(NZ)-S64(OG), K67(NZ)-N152(OD1), and K67(NZ)-G220(O) are formed in wild-type PDC-3 (orange) but broken in the E219K (pink) and Y221A (blue) variants. (C) pH titration curves for K67 based on three replicate constant-pH MD simulations. Each point indicates the fraction of deprotonated K67 at a given pH, and the lines are best fits to a titration model. The estimated pKa values are shown in the legend.
The fraction (%) of the formation of critical hydrogen bonds in the active sites.
Structural visualization of the enlarged active-site pockets.
(A) The K67(NZ)-G220(O), Y150(N)-A292(O), and N287(ND2)-N314(OD1) interactions in structures from wild-type PDC-3 (orange), V211A (green), and Y221A (blue) variants. The yellow dashed lines represent the interactions. (B) The active site pockets of wild-type PDC-3 (orange), V211A (green), and Y221A (blue) variants are shown as surface representation.
Mean (± Standard Deviation) pocket volume (Å3) and solvent accessible surface area (Å2) in the active-site pocket for wild-type (WT) and its variants.
T-statistics and p-values were derived from two-sample t-tests comparing each mutant with WT. p-values below 0.0001 are reported as ‘p < 0.0001’.
Correlation coefficients between the distances of key interactions (32 features) and volume of active-site pocket.
Free energy landscapes and metastable-state distributions from Markov State Models reveal key conformational transitions in wild-type PDC-3 and its variants.
(A) The free energy landscape for the microstates of the wild-type PDC-3 and its mutants. (B) The metastable states grouped from microstates of PDC-3 and its variants systems. The microstates were grouped by the PCCA method into metastable states in all systems.
The distribution of RMSD values of the wild-type PDC-3 and its variants.
The three violin plots illustrate the RMSD values of the complete protein, the Ω-loops, and the R2-loops, respectively. The width of each violin plot represents the density of data points at a given RMSD value. The median and quartiles are indicated by the white dot and the thick black line inside the violin plot, respectively. The crystal structure PDB ID 4HEF is used as the reference.
RMSD as a function of the fraction of the atoms considered in the alignment.
The distance distributions of the tridentate hydrogen-bonding interactions (K67(NZ)-S64(OG), K67(NZ)-N152(OD1) and K67(NZ)-G220(O).
Each violin represents the probability density of observed distances, with median and quartiles indicated by the white dot and the thick black line inside the violin plot, respectively.
Time-resolved deprotonation of K67 in WT, E219K, and Y221A over 200-ns constant-pH MD simulations at six pH values (5, 6, 7, 8, 9, 10, and 11).
Each panel plots the deprotonated fraction of K67 versus simulation time for one replica.
The distance distributions of the three key hydrogen-bonding interactions (K67(NZ)-G220(O), Y150(N)-A292(O), and N287(ND2)-N314(OD1)).
Each violin represents the probability density of observed distances, with median and quartiles indicated by the white dot and the thick black line inside the violin plot, respectively.
The distribution of pocket volume and solvent accessible surface area for the wild-type PDC3 enzyme and nine mutants.
Each violin represents the probability density of observed distances, with median and quartiles indicated by the white dot and the thick black line inside the violin plot, respectively.
Docking of ceftolozane into the active-site pockets of the wild-type PDC-3, V211A and Y221A variants.
The convergence behaviour of the implied timescales related to the first ten slowest processes.
The Chapman Kolmogorov test plot of wild-type PDC-3.
The Chapman Kolmogorov test plot of V211A variant.
The Chapman Kolmogorov test plot of V211G variant.
The Chapman Kolmogorov test plot of G214A variant.
The Chapman Kolmogorov test plot of G214R variant.
The Chapman Kolmogorov test plot of E219A variant.
The Chapman Kolmogorov test plot of E219G variant.
The Chapman Kolmogorov test plot of E219K variant.
The Chapman Kolmogorov test plot of Y221A variant.
The Chapman Kolmogorov test plot of Y221H variant.
TICA plot illustrates the distribution of wild-type PDC-3 and its variants with the colour indicating the K67(NZ)-S64(OG) distance.
TICA plot illustrates the distribution of wild-type PDC-3 and its variants with the colour indicating the K67(NZ)-N152(OD1 distance.
TICA plot illustrates the distribution of wild-type PDC-3 and its variants with the colour indicating the K67(NZ)-G220(O) distance.
TICA plot illustrates the distribution of wild-type PDC-3 and its variants with the colour indicating the Y150(N)-A292(O) distance.
