Remodeling of the Binding Site of Nucleoside Diphosphate Kinase Revealed by X-ray Structure and H/D Exchange

Abstract

To be fully active and participate in the metabolism of phosphorylated nucleotides, most nucleoside diphosphate kinases (NDPKs) have to assemble into stable hexamers. Here we studied the role played by six intersubunit salt bridges R80–D93 in the stability of NDPK from the pathogen Mycobacterium tuberculosis (Mt). Mutating R80 into Ala or Asn abolished the salt bridges. Unexpectedly, compensatory stabilizing mechanisms appeared for R80A and R80N mutants and we studied them by biochemical and structural methods. The R80A mutant crystallized into space group I222 that is unusual for NDPK, and its hexameric structure revealed the occurrence at the trimer interface of a stabilizing hydrophobic patch around the mutation. Functionally relevant, a trimer of the R80A hexamer showed a remodeling of the binding site. In this conformation, the cleft of the active site is more open, and then active His117 is more accessible to substrates. H/D exchange mass spectrometry analysis of the wild type and the R80A and R80N mutants showed that the remodeled region of the protein is highly solvent accessible, indicating that equilibrium between open and closed conformations is possible. We propose that such equilibrium occurs in vivo and explains how bulky substrates access the catalytic His117.

Structure, Folding and Stability of Nucleoside Diphosphate Kinases

“…Importantly, hydrogen deuterium exchange mass spectrometry (HDX-MS) revealed that residues 40–70 are the only ones inside the hexamer that are fully solvent accessible, indicating that an equilibrium between these two conformations is possible ( Figure 3 C,F). Such behavior was also noticed for the WT (wild type) as well as for two point-mutants (R80A; R80N) for which the mutation site is not part of the remodeled region of the protein [ 41 ]. It is tempting to speculate that such equilibrium occurs in vivo, explaining how bulky substrates (e.g., nucleic acids or peptides) access the catalytic His117.…”

“…Together, these studies underscore the critical role of quaternary structure in the stability of NDPK and showed that during evolution, different stabilization mechanisms can develop inside the same family of proteins. mechanism was discovered by analyzing the structure of R80A mutant, which also shows little destabilization (Tm of 69 °C), despite the disappearance of ionic bonds [41]. In this case, stabilization was generated through the formation of a hydrophobic patch including the hydrophobic Ala93 (Figure 4D).…”

“…ADP is bound in the active site by homology with NDPK-A. (Figure adapted with permission from [ 41 ]).…”

“…Recently, an important remodeling has been revealed for one trimer inside the NDPK hexamer from M. tuberculosis ( Figure 3 D–F), the second trimer presenting the classical unmodified conformation ( Figure 3 A–C) [ 41 ]. The remodeling was identical for all three subunits of the trimer, covering residues 40–70 and contributing to: (i) the full disappearance of α A -α 2 helix hairpin; (ii) the appearance of a new structure formed by successive β-turns of type I and II covering residues 58–66; (iii) the increase of lengths for strands β 2 and β 3 ; and (iv) the absence of stable structure for the region covering residues 48–57 that are no longer visible in the electron densities.…”

“…The 1.9 Å resolution of the R80N mutant structure provided the mechanism: the side chain of N80 forms two new strong hydrogen bonds with two neighboring subunits ( Figure 4 C) [ 28 ]. Another stabilizing mechanism was discovered by analyzing the structure of R80A mutant, which also shows little destabilization (T m of 69 °C), despite the disappearance of ionic bonds [ 41 ]. In this case, stabilization was generated through the formation of a hydrophobic patch including the hydrophobic Ala93 ( Figure 4 D).…”