Fungal cobalamin-independent methionine synthase: Insights from the model organism, Neurospora crassa.

Two families of methionine synthases, distinct in catalytic and structural features, have been encountered: MetH, the cobalamin-dependent enzyme and MetE, the cobalamin-independent form. The MetE family is of mechanistic interest due to the chemically challenging nature of the reaction and is a potential target for antifungal therapeutics since the human genome encodes only MetH. Here we report the identification, purification, and crystal structure of MetE from the filamentous fungus Neurospora crassa (ncMetE). ncMetE was highly thermostable and crystallized readily, making it ideal for study. Crystal structures of native ncMetE in complex with either Zn(2+)or Cd(2+) were solved at resolution limits of 2.10 Å and 1.88 Å, respectively. The monomeric protein contains two domains, each containing a (ßα)8 barrel core, and a long α-helical segment spans the length of the protein, connecting the domains. Zn(2+) bound in the C-terminal domain exhibits tetrahedral coordination with the side chains of His 652, Cys 654, Glu 676 and Cys 737. A Cd(2+) replete structure revealed a supermetalated enzyme and demonstrated the inate flexibility of the metal binding site. An extensive analysis of sequence conservation within the MetE family identified 57 highly conserved residues and 60 additional residues that were conserved in all fungal sequences examined.

In silico identification and analysis of the binding site for aminocoumarin type inhibitors in the C-terminal domain of Hsp90.

Hsp90 contains two Nucleotide Binding Sites (NBS): one each in its N-terminal domain (NTD) and C-terminal domain (CTD), respectively. Previously we used computational techniques to locate a nucleotide-binding site in the CTD. Nucleotide binding at this site stabilized the structurally labile region within this domain, thus providing a rationale for increased resistance to thermal denaturation and proteolysis. A scan for ligand-binding sites in CTD revealed four potential sites with the requisite volume to accommodate aminocoumarins and -derived inhibitors. Only one of these reproducibly formed docked complexes with inhibitors and showed excellent interactions with residues lining the site. Fortuitously, it was identical to the aforementioned nucleotide-binding site thus providing an explanation for the reported direct competition between inhibitors and nucleotides. Further studies with carefully chosen inhibitors and some inactive analogues provided an explanation for the known Structure-Activity Relationships (SAR) of aminocoumarin and -derived inhibitors. We also performed similar studies of the NTD to discern the reason(s) for its inability to bind aminocoumarins, given the family resemblance to prokaryotic Top-IV and Gyr-B. Our studies permitted the identification of the putative inhibitor binding site in the CTD, an explanation for increased resistance to thermal denaturation and proteolysis upon inhibitor binding as well as direct competition with ATP.

In silico identification and computational analysis of the nucleotide binding site in the C-terminal domain of Hsp90.

Hsp90 contains two distinct Nucleotide Binding Sites (NBS), in its N-terminal domain (NTD) and C-terminal domain (CTD), respectively. The NTD site belongs to the GHKL super-family of ATPases and has been the subject of extensive characterization. However, a structure of the nucleotide-bound form of CTD is still unavailable. In this study molecular modeling was employed to incorporate experimental data using partial constructs of the CTD, from work published by many research groups, onto existing structural models of its apo- form. Our attempts to locate potential nucleotide ligand-binding sites or cavities yielded one major candidate-a structurally unconventional site-exhibiting the requisite shape and volume for accommodation of tri-phosphate nucleotides. Its structure was refined by molecular dynamics (MD)-based techniques. We reproducibly docked the Mg2+ complexed form of ATP, GTP, CTP, TTP and UTP to this putative NBS. These docking simulations and calculated ligand-binding scores are in general agreement with published data about experimentally measured binding to the CTD. The overall pattern of interactions between residues lining the site and docked nucleotides is conserved and broadly similar to that of other nucleotide-binding sites. Our docking simulations suggest that nucleotide binding stabilizes the only structurally labile region, thereby providing a rationale for the increased resistance to thermal denaturation and proteolysis. The docked nucleotides do not intrude onto the surface of residues involved in dimerization or chaperoning. Our molecular modeling permitted recognition of larger structural changes in the nucleotide-bound CTD dimer, including stabilization of helix-2 in both chains and intra- and inter- chain interactions between three residues (I613, Q617, R620).

Homology modeling, ligand docking and in silico mutagenesis of neurospora Hsp80 (90): insight into intrinsic ATPase activity.

The Hsp90 family of proteins is an important component of the cellular response to elevated temperatures, environmental or physiological stress and nuclear receptor signalling. The primary object of this work is the 80-kDa heat shock protein, a member of the Hsp90 family, from the model filamentous fungus Neurospora crassa, (henceforth referred to as Hsp80Nc). In contrast to more extensively characterized members of the same family, (e.g. Hsp82Sc of Saccharomyces cerevisiae) it exhibits a higher intrinsic ATPase activity and the ability to form hetero-oligomeric complexes with Hsp70 in the absence of co-chaperones or other ancillary factors. As unabridged experimentally derived structures of Hsp80Nc or Hsp82Sc are not available; we developed homology-based models for both of them. A structural analysis and comparison of these models was undertaken to better understand the nature of dimerization-induced changes in secondary structure and patterns of residue interaction. Our studies yielded some interesting and novel insights into the synergistic and mutually reinforcing nature of interactions between major domains of the two chains in their dimeric forms. We also evaluated the effect of residue substitutions in the 'lid' region of Hsp80Nc and Hsp82Sc on the calculated ligand-binding energy of ATP (and ADP) to their respective N-terminal domains. Our studies suggest that the higher intrinsic ATPase activity of Hsp80Nc may be attributable to differences in the residue sequences between the lid region of these two proteins.