Solution‐state structure by NMR of zinc‐substituted rubredoxin from the marine hyperthermophilic archaebacterium pyrococcus furiosus

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Abstract
The three‐dimensional solution‐state structure is reported for the zinc‐substituted form of rubredoxin (Rd) from the marine hyperthermophilic archaebacterium Pyrococcus furiosus, an organism that grows optimally at 100°C. Structures were generated with DSPACE by a hybrid distance geometry (DG)‐based simulated annealing (SA) approach that employed 403 nuclear Overhauser effect (NOE)‐derived interproton distance restraints, including 67 interresidue, 124 sequential (i – j = 1), 75 medium‐range (i – j = 2–5), and 137 long‐range (i – j > 5) restraints. All lower interproton distance bounds were set at the sum of the van Der Waals radii (1.8 å), and upper bounds of 2.7 å, 3.3 å, and 5.0 å were employed to represent qualitatively observed strong, medium, and weak NOE cross peak intensities, respectively. Twenty‐three backbone‐backbone, six backbone‐sulfur (Cys), two backbone‐side chain, and two side chain‐side chain hydrogen bond restraints were include for structure refinement, yielding a total of 436 nonbonded restraints, which averages to > 16 restraints per residue. A total of 10 structures generated from random atom positions and 30 structures generated by molecular replacement using the backbone coordinates of Clostridium pasteurianum Rd converged to a common conformation, with the average penalty (= sum of the square of the distance bounds violations; ± standard deviation) of 0.024 ± 0.003 å2 and a maximum total penalty of 0.035 å2. Superposition of the backbone atoms (C, Cα, N) of residues A1‐L51 for all 40 structures afforded an average pairwise root mean square (rms) deviation value (±SD) of 0.42 ± 0.07 å. Superposition of all heavy atoms for residues A1‐L51, including those of structurally undefined external side chains, afforded an average pairwise rms deviation of 0.72 ± 0.08 å. Qualitative comparison of back‐calculated and experimental two‐dimensional NOESY spectra indicate that the DG/SA structures are consistent with the experimental spectra. The global folding of P. furiosus Zn(Rd) is remarkably similar to the folding observed by X‐ray crystallography for native Rd from the mesophilic organism C. pasteurianum, with the average rms deviation value for backbone atoms of residues A1‐L51 of P. furiosus Zn(Rd) superposed with respect to residues K2–V52 of C. pasteurianum Rd of 0.77 ± 0.06 å. The conformations of aromatic residues that compose the hydrophobic cores of the two proteins are also similar. However, P. furiosus Rd contains several unique structural elements, including at least four additional hydrogen bonds and three potential electrostatic interactions. Four of these interactions involve the nonconservatively substituted Glu 14, Ala 1, and Trp 3 residues. The combined findings are consistent with the proposal that stabilization of the N‐terminal residues inhibits the β‐sheet from “unzipping” at elevated temperatures (Blake, P.R., Park, J.‐B., Bryant, F.O., et al., 1991, Biochemistry 30, 10885–10895). In view of the high structural similarities between this hyperthermophilic protein and C. pasteurianum Rd, this effect may serve as the dominant mechanism by which P. furiosus Rd is stabilized at high temperatures.