Ω-Loop mutations control dynamics of the active site by modulating the hydrogen-bonding network in PDC-3 β-lactamase
- University College London, United Kingdom
- Research Service, Louis Stokes Cleveland, Department of Veterans Affairs Medical Center, United States
- Department of Molecular Biology and Microbiology, Case Western Reserve University School of Medicine, United States
- Department of Medicine, Case Western Reserve University School of Medicine, United States
- Clinician Scientist Investigator, Louis Stokes Cleveland Department of Veterans Affairs Medical Center, United States
- Departments of Pharmacology, Biochemistry, and Proteomics and Bioinformatics, Case Western Reserve University School of Medicine, United States
- CWRU-Cleveland VAMC Center for Antimicrobial Resistance and Epidemiology (Case VA CARES) Cleveland, United States
- UCL Centre for Advanced Research Computing, United Kingdom
- University of Tabuk (PFSCBR), Saudi Arabia
Figures
Figure 1
Structures and catalytic mechanism of -lactam antibiotics and PDC-3 -lactamase.
(A) Structures of representative -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 -lactamase hydrolysis of cephalosporins. (D) The overall structure of the protein is shown in a cartoon representation (PDB ID: 4HEF). The -loop and R2-loop are colored orange and red, respectively. The conserved residues in the active site are colored green and highlighted as sticks. The positions of all mutations (V211A/G, G214A/R, E219A/G/K, and Y221A/H) are highlighted as red spheres.
Figure 2 with 2 supplements
Structural stability and dynamic flexibility analyses of wild-type PDC-3 -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. (B) The root-mean-square fluctuation (RMSF) of wild-type PDC-3 and its variants. The -loop (residues G183 to S226) is highlighted in yellow, and the R2-loop (residues L280 to Q310) is highlighted in blue. (C) Core Cα RMSD superimposition of wild-type 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%).
Figure 2—figure supplement 1
The distribution of RMSD values of wild-type PDC-3 and its variants.
The three violin plots illustrate the RMSD values of the complete protein, the -loop, and the R2-loop, 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.
Figure 2—figure supplement 2
RMSD as a function of the fraction of the atoms considered in the alignment.
Figure 3 with 12 supplements
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.
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Figure 3—source data 1
The mean first passage time (MFPT) estimates.
- https://cdn.elifesciences.org/articles/107688/elife-107688-fig3-data1-v1.docx
Figure 3—figure supplement 1
Correlation coefficients between the distances of key interactions (32 features) and volume of active-site pockets.
Figure 3—figure supplement 2
The convergence behavior of the implied timescales related to the first 10 slowest processes.
Figure 3—figure supplement 3
The Chapman–Kolmogorov test plot of wild-type PDC-3.
Figure 3—figure supplement 4
The Chapman–Kolmogorov test plot of V211A variant.
Figure 3—figure supplement 5
The Chapman–Kolmogorov test plot of V211G variant.
Figure 3—figure supplement 6
The Chapman–Kolmogorov test plot of G214A variant.
Figure 3—figure supplement 7
The Chapman–Kolmogorov test plot of G214R variant.
Figure 3—figure supplement 8
The Chapman–Kolmogorov test plot of E219A variant.
Figure 3—figure supplement 9
The Chapman–Kolmogorov test plot of E219G variant.
Figure 3—figure supplement 10
The Chapman–Kolmogorov test plot of E219K variant.
Figure 3—figure supplement 11
The Chapman–Kolmogorov test plot of Y221A variant.
Figure 3—figure supplement 12
The Chapman–Kolmogorov test plot of Y221H variant.
Figure 4 with 4 supplements
E219K and Y221A mutations reshape the catalytic conformations and protonation states of K67.
(A) 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. (B) The 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 values are shown in the legend.
Figure 4—figure supplement 1
TICA plot illustrates the distribution of wild-type PDC-3 and its variants with the color indicating the K67(NZ)-S64(OG) distance.
Figure 4—figure supplement 2
TICA plot illustrates the distribution of wild-type PDC-3 and its variants with the color indicating the K67(NZ)-N152(OD1) distance.
Figure 4—figure supplement 3
TICA plot illustrates the distribution of wild-type PDC-3 and its variants with the color indicating the K67(NZ)-G220(O) distance.
Figure 4—figure supplement 4
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.
Figure 5 with 2 supplements
Structural visualization of the enlarged active-site pocket.
(A) The K67(NZ)-G220(O), Y150(N)-A292(O), and N287(ND2)-N314(OD1) interactions in representative structures of wild-type PDC-3, Y221A, and V211A variants. The yellow dashed lines represent the interactions. (B) The active-site pockets of wild-type PDC-3, Y221A, and V211A variants are shown as surface representation.
Figure 5—figure supplement 1
TICA plot illustrates the distribution of wild-type PDC-3 and its variants with the color indicating the Y150(N)-A292(O) distance.
Figure 5—figure supplement 2
TICA plot illustrates the distribution of wild-type PDC-3 and its variants with the color indicating the N287(ND2)-N314(OD1) distance.
Figure 6 with 1 supplement
Metastable-state pocket volumes and key interaction patterns across wild-type PDC-3 and its variants.
(A) Mean active-site pocket volume for each metastable state in wild-type PDC-3 and its variants. Bars denote the mean pocket volume (Å3), and error bars indicate the standard deviation across frames assigned to each state. (B) Heat maps show the mean distances (Å) for three contacts that may contribute to pocket-volume changes across MSM metastable states (K67(NZ)–G220(O), Y150(N)–A292(O), and N287(ND2)–N314(OD1)). Color encodes the distance magnitude, with blue indicating lower values and red indicating higher values.
Figure 6—figure supplement 1
Mean distances of three contacts (K67(NZ)–G220(O), Y150(N)–A292(O), and N287(ND2)–N314(OD1)) across metastable states in wild-type PDC-3 and its variants.
Bars denote the mean donor–acceptor distance (Å) for each contact within each state, and error bars indicate the standard deviation across frames assigned to that state.
Author response image 1
TICA plot illustrates the distribution of E219K with the colour indicating the K67(NZ)-S64(OG), K67(NZ)-N152(OD1) and K67(NZ)-G220(O) distance.
Author response image 2
The root-mean-square fluctuation (RMSF) profiles of wild-type PDC-3 and its variants.
Blue lines show the mean RMSF across 100 independent MD trajectories for each system; red translucent bands denote the standard deviation across trajectories. The Ω-loop (residues G183 to S226) is highlighted in yellow, and the R2-loop (residues L280 to Q310) is highlighted in blue.
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Ω-Loop mutations control dynamics of the active site by modulating the hydrogen-bonding network in PDC-3 β-lactamase
eLife 14:RP107688.
https://doi.org/10.7554/eLife.107688.3
