Solution NMR refinement of a metal ion bound protein using metal ion inclusive restrained molecular dynamics methods

Abstract

Correctly calculating the structure of metal coordination sites in a protein during the process of nuclear magnetic resonance (NMR) structure determination and refinement continues to be a challenging task. In this study, we present an accurate and convenient means by which to include metal ions in the NMR structure determination process using molecular dynamics (MD) simulations constrained by NMR-derived data to obtain a realistic and physically viable description of the metal binding site(s). This method provides the framework to accurately portray the metal ions and its binding residues in a pseudo-bond or dummy-cation like approach, and is validated by quantum mechanical/molecular mechanical (QM/MM) MD calculations constrained by NMR-derived data. To illustrate this approach, we refine the zinc coordination complex structure of the zinc sensing transcriptional repressor protein Staphylococcus aureus CzrA, generating over 130 ns of MD and QM/MM MD NMR-data compliant sampling. In addition to refining the first coordination shell structure of the Zn(II) ion, this protocol benefits from being performed in a periodically replicated solvation environment including long-range electrostatics. We determine that unrestrained (not based on NMR data) MD simulations correlated to the NMR data in a time-averaged ensemble. The accurate solution structure ensemble of the metal-bound protein accurately describes the role of conformational sampling in allosteric regulation of DNA binding by zinc and serves to validate our previous unrestrained MD simulations of CzrA. This methodology has potentially broad applicability in the structure determination of metal ion bound proteins, protein folding and metal template protein-design studies.

Fig. 1

Calculated structural ensembles of Zn(II)-bound CzrA from NMR structure determination while including the metal ion (panels a-c) and without including the metal ion (panels d-f). (a) Ribbon representation of ten metal ion refined NMR structures of Zn(II)-CzrA from independent QM/MM MD calculations. Zn(II) ions are shown as silver spheres and the zinc binding residues (Asp84, His86, His97' and His100') are depicted in licorice notation. (b) The zinc ion coordination in a random model selected from the calculated QM/MM MD NMR ensemble of structures. (c) An overlay of metal-binding residue side-chain atoms at a metal binding site for the calculated QM/MM MD NMR ensemble of structures. In a similar manner, panel (d) shows a ribbon representation for the 2KJC ensemble of NMR structures. (e) Arrangement of zinc binding residues at a metal binding site for a random model for the 2KJC structural ensemble. (f) An overlay of zinc binding residues for the 2KJC structural ensemble. The zinc binding residues in panels (a) and (d) are presented from a different angle compared to panels (b), (c), (e) and (f). The backbone atoms of Asp84, His86, His97' and His100' were aligned to create figures (c) and (f)

Fig. 2

(a) Correlation of experimental backbone NH (¹D_NH) RDC constraints with those calculated from (a) the 1R1V crystal structure of Zn-bound CzrA (R = 0.944; y = 1.002x + 0.036), (b) the average NMR solution structure for the 2KJC ensemble (R = 0.988; y = 0.999x − 0.078), (c) model 1 of the 2KJC ensemble of NMR structures before (R = 0.934; y = 0.994x − 0.178) and (d) after the metal ion refinement MRD-NMR procedure described here (R = 0.999; y = 0.997x + 0.017). R is the correlation coefficient and the equation of the fitted line is in the form of y = mx +c where, ‘m’ defines the slope of the line and ‘c’ the value of x at y=0

Fig. 3

(a) Normalized population distribution of the Bax regression slopes(Zweckstetter and Bax 2000) indicating the quality of fitting between the experimental backbone NH(¹D_NH) RDC constraints with those calculated for ensembles of structures from NOE+RDC-restrained MD and QM/MD simulations (MRD-NMR structural ensemble), NOE-restrained MD simulations and unrestrained MD simulations of Zn(II)-CzrA. A higher regression slope indicates a better fit to the data. (b) Normalized population distributions of calculated NH(¹D_NH) RDC-deviations from experimentally determined values for the ensemble of structures from unrestrained MD simulations, NOE restrained MD simulations, and NOE + RDC restrained MD simulations of Zn(II)-CzrA

Fig. 4

(a) The zinc mediated His97-His67' hydrogen bond that forms a part of the hydrogen-bonding pathway in Zn(II)-CzrA. The zinc ion is shown as a silver sphere while His97 and His67' residues are in licorice notation. The His97-His67' hydrogen bond is indicated with a dotted black line. (b) Normalized population distribution of heavy atom hydrogen bond distances (Nε-O) for the 2KJC ensemble of structures and the MRD-NMR ensemble of structures. The crystallographically determined His97-His67' distance is shown for comparison

Fig. 5

Ribbon representation of (a) the 1R1V crystal structure of Zn(II)-CzrA and (b) a MRD-NMR refined structure of Zn(II)-CzrA near the metal binding site. Interactions between Asp83 and Lys70 (blue dotted line) and Asp83 and Leu68 (red dotted line) are indicated. (c) Normalized population distributions of the heavy atom hydrogen bond distances for the metal ion refined RDC+NOE constrained MD and QM/MM MD ensemble of structures

Fig. 6

(a) Ribbon representation of the MRD-NMR structure of zinc bound CzrA indicating residues used to measure changes between the open and closed conformations. Zinc ions are shown as silver spheres and protein residues are shown in licorice depiction. Residues involved in metal ion binding are colored lilac. Normalized population distributions of (b) Ser54-Ser54' and (c) Gly75-Gly75' inter-subunit Cα-Cα distances and (d) Ser54-Ser65-Ser54' and Ser54'-Ser65'-Ser54 inter-subunit Cα-Cα-Cα angles for the 2KJC ensemble of NMR structures of Zn(II)-CzrA, the 2KJB ensemble of NMR structures of DNA-bound CzrA, and the metal ion refined RDC+NOE constrained MD and QM/MM MD ensemble of Zn(II)-CzrA structures. Distances from the 1R1V Zn(II)-bound crystal structure of CzrA are also indicated