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. 2019 Apr 30;9(1):6656.
doi: 10.1038/s41598-019-42866-8.

The Structural Dynamics of Engineered β-Lactamases Vary Broadly on Three Timescales Yet Sustain Native Function

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Free PMC article

The Structural Dynamics of Engineered β-Lactamases Vary Broadly on Three Timescales Yet Sustain Native Function

Sophie M C Gobeil et al. Sci Rep. .
Free PMC article

Abstract

Understanding the principles of protein dynamics will help guide engineering of protein function: altering protein motions may be a barrier to success or may be an enabling tool for protein engineering. The impact of dynamics on protein function is typically reported over a fraction of the full scope of motional timescales. If motional patterns vary significantly at different timescales, then only by monitoring motions broadly will we understand the impact of protein dynamics on engineering functional proteins. Using an integrative approach combining experimental and in silico methodologies, we elucidate protein dynamics over the entire span of fast to slow timescales (ps to ms) for a laboratory-engineered system composed of five interrelated β-lactamases: two natural homologs and three laboratory-recombined variants. Fast (ps-ns) and intermediate (ns-µs) dynamics were mostly conserved. However, slow motions (µs-ms) were few and conserved in the natural homologs yet were numerous and widely dispersed in their recombinants. Nonetheless, modified slow dynamics were functionally tolerated. Crystallographic B-factors from high-resolution X-ray structures were partly predictive of the conserved motions but not of the new slow motions captured in our solution studies. Our inspection of protein dynamics over a continuous range of timescales vividly illustrates the complexity of dynamic impacts of protein engineering as well as the functional tolerance of an engineered enzyme system to new slow motions.

Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Hybrid active sites of the β-lactamase chimeras under investigation. (a) The native class A TEM-1 and PSE-4 β-lactamases (40% sequence identity) were recombined to yield chimeras,. Segments originating from TEM-1 (blue) and PSE-4 (green) in the chimeras cTEM-2m, cTEM-17m, cTEM-19m; ‘c’ indicates chimera and ‘m’ the number of substitutions relative to TEM-1. Deconvolution of the mutations at positions 68 and 69 gave cTEM-18m(M68L), cTEM-18m(M69T), TEM-1(M68L) and TEM-1(M69T). Numbering according to Ambler. The catalytic nucleophile (Ser70) and Ω-loop are indicated. (b) Structural representation of cTEM-2m (PDB ID: 4MEZ) and cTEM-19m (PDB ID: 4R4S), colored as in (a), highlight the hybrid active site composition at the interface of the all-α and α/β domains. (c) Active-site walls, set in TEM-1 (PDB ID: 1XPB). Green: S70 wall (Met69-Lys73); lilac, Y105 wall (Val 103-Ser106); dark blue, SDN wall (Met129-Asn132); red, Ω-loop wall (Glu166-Asn170); orange, 214–218 wall; and cyan, 234–244 wall. (Right) Solvent-accessible surface of the active-site walls. (d) Reaction scheme for the hydrolysis of β-lactams by β-lactamases.
Figure 2
Figure 2
Crystal structures of cTEM-2m and cTEM-19m. (a) Backbone overlay of chimeras cTEM-2m (red; PDB ID: 4MEZ) and cTEM-19m (orange; PDB ID: 4R4S) over TEM-1 (blue; PDB ID: 1XPB). (b) Active-site view of (a). Wat: new water molecule in cTEM-2m (Wat519) and cTEM-19m (Wat478) relative to PSE-4 (green; Wat483). Tyr105 of cTEM-19m was modeled to the m-30° conformation, as opposed to the t80° conformation observed in TEM-1 and cTEM-2m. (c) Overlay of the active-site volume of TEM-1 and cTEM-2m. Orientation as in Fig. 1c. (d) Overlay of the active-site volume of TEM-1, cTEM-17m (yellow; PDB ID: 4ID4) and cTEM-19m. For residues with alternate conformations in the crystal structure, the conformer with the highest occupancy was illustrated. See Table S2 for data collection and refinement statistics.
Figure 3
Figure 3
β-Lactamase dynamics on the ps to ms timescales. Dynamics of the parents TEM-1 (blue, PDB ID: 1XPB), PSE-4 (green, PDB ID: 1G68) and the laboratory-evolved chimeras cTEM-2m (PDB ID: 4MEZ), cTEM-17m (PDB ID: 4ID4) and cTEM-19m (PDB ID: 4R4S) colored blue/green according to the parental origin of each segment, as in Fig. 1. Left: Dynamic residues on the ps-ns timescale are given by the MD-derived S2 order parameter for the amide NH. Residues with S2 lower than protein average S2 are colored and thickened on a scale of yellow (<0.85; less dynamic) to red (≤0.7; more dynamic). Middle: Dynamics on the ns-µs timescale are given by the Cα-RMSF calculated from triplicate 2 µs MD simulations. Residues with Cα-RMSF above the protein average Cα-RMSF are colored and thickened on a scale of yellow (>0.10 nm; less dynamic) to bright red (≥0.25 nm; more dynamic). Right: Dynamics on the µs-ms timescale were monitored by CPMG-NMR on the 1H-15N vector. Residues showing dispersion curves with Δ R2 ≥ 7.0 s-1 at 800 MHz are colored orange. NMR unassigned residues are colored gray. Raw data are presented in Tables S2 to S7 and Figs S1 to S4.
Figure 4
Figure 4
Global exchange rates (kex) of cTEM-2m, cTEM-17m and cTEM-19m. The kex of the fitted regions are colored and thickened on a scale of yellow (300 s−1) to red (1500 s−1) on the crystal structure coordinates of cTEM-2m (PDB ID: 4MEZ), cTEM-17m (PDB ID: 4ID4) and cTEM-19m (PDB ID: 4R4S). NMR unassigned residues are colored dark gray. The fitted regions were the N-terminal helix (H1, residues 28–41), S70 wall (residues 69–72), Y105 wall (residues 105–106), SDN wall (residues 128–132), Ω-loop wall (residues 166–170), the domain connector (214–225), 234–244 wall and the C-terminal helix (H11, 272–288).
Figure 5
Figure 5
Distribution of motions in the β-lactamases over the 3 observed timescales in relation to the linear sequence and crystallographic B-factors. The sequence blocks originating from TEM-1 (blue) and PSE-4 (green) are colored in cTEM-2m, cTEM-17m and cTEM-19m according to their parental origin as in Fig. 1. Crystallographic B-factors (B) above the protein average are colored on a scale of yellow (average + 1 st. dev.) to bright red (highest B-factor for that protein) (PDBs 1G68, 1XPB, 4MEZ, 4ID4 and 4R4S, respectively). The ps to ms motions are shown in yellow (less dynamic) to red (more dynamic), as in Fig. 3. NMR unassigned residues are colored gray.

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References

    1. Ramanathan A, Savol A, Burger V, Chennubhotla CS, Agarwal PK. Protein conformational populations and functionally relevant substates. Acc Chem Res. 2014;47:149–156. doi: 10.1021/ar400084s. - DOI - PubMed
    1. Henzler-Wildman K, Kern D. Dynamic personalities of proteins. Nature. 2007;450:964–972. doi: 10.1038/nature06522. - DOI - PubMed
    1. Bhabha G, et al. A dynamic knockout reveals that conformational fluctuations influence the chemical step of enzyme catalysis. Science. 2011;332:234–238. doi: 10.1126/science.1198542. - DOI - PMC - PubMed
    1. Boehr DD, McElheny D, Dyson HJ, Wright PE. Millisecond timescale fluctuations in dihydrofolate reductase are exquisitely sensitive to the bound ligands. Proc Natl Acad Sci USA. 2010;107:1373–1378. doi: 10.1073/pnas.0914163107. - DOI - PMC - PubMed
    1. Gagné D, et al. Perturbation of the Conformational Dynamics of an Active-Site Loop Alters Enzyme Activity. Structure. 2015;23:2256–2266. doi: 10.1016/j.str.2015.10.011. - DOI - PMC - PubMed

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