Isocitrate dehydrogenase 1 gene variants analysis of glioma patients from Pakistan.

Various somatic isocitrate dehydrogenase 1 (IDH1) gene variants have been reported to drive lower-grade gliomas and secondary glioblastomas. In the current study, we explored the IDH1 variants in the glioma biopsy samples of patients from Pakistan. We explored the incidence of isocitrate dehydrogenase 1 gene variants by hotspot sequencing in 80 formalin-fixed paraffin-embedded tissues of different types of glioma biopsy samples. Structural modeling of the identified variants in isocitrate dehydrogenase 1 protein was done to see their possible consequences. The frequently described p.Arg132 variants were not found in any of the glioma types. However, in our study, we identified nonsynonymous variants at the residues p.R109 and p.G136 in astrocytomas and p.R100 in oligodendroglioma. These variants are affecting a part of the conserved domain in isocitrate dehydrogenase 1. Both of p.R100 and p.R109 variants are rare and described before, whereas the p.G136 variant identified in this study has never been described previously. Structural modeling showed that variants of these residues would directly affect the substrate binding and hence the enzyme activity.

Molecular mechanism of the dual regulatory roles of ATP on the αγ heterodimer of human NAD-dependent isocitrate dehydrogenase.

Human NAD-dependent isocitrate dehydrogenase (NAD-IDH) is responsible for the catalytic conversion of isocitrate into α-ketoglutarate in the Krebs cycle. This enzyme exists as the α2ßγ heterotetramer composed of the αß and αγ heterodimers. Our previous biochemical data showed that the αγ heterodimer and the holoenzyme can be activated by low concentrations of ATP but inhibited by high concentrations of ATP; however, the molecular mechanism was unknown. Here, we report the crystal structures of the αγ heterodimer with ATP binding only to the allosteric site (αMgγMg+CIT+ATP) and to both the allosteric site and the active site (αMg+ATPγMg+CIT+ATP). Structural data show that ATP at low concentrations can mimic ADP to bind to the allosteric site, which stabilizes CIT binding and leads the enzyme to adopt an active conformation, revealing why the enzyme can be activated by low concentrations of ATP. On the other hand, at high concentrations ATP is competitive with NAD for binding to the catalytic site. In addition, our biochemical data show that high concentrations of ATP promote the formation of metal ion-ATP chelates. This reduces the concentration of free metal ion available for the catalytic reaction, and thus further inhibits the enzymatic activity. The combination of these two effects accounts for the inhibition of the enzyme at high concentrations of ATP. Taken together, our structural and biochemical data reveal the molecular mechanism for the dual regulatory roles of ATP on the αγ heterodimer of human NAD-IDH.

Molecular basis for the function of the αß heterodimer of human NAD-dependent isocitrate dehydrogenase.

Mammalian mitochondrial NAD-dependent isocitrate dehydrogenase (NAD-IDH) catalyzes the decarboxylation of isocitrate into α-ketoglutarate in the tricarboxylic acid cycle. It exists as the α2ßγ heterotetramer composed of the αß and αγ heterodimers. Different from the αγ heterodimer that can be allosterically activated by CIT and ADP, the αß heterodimer cannot be allosterically regulated by the activators; however, the molecular mechanism is unclear. We report here the crystal structures of the αß heterodimer of human NAD-IDH with the α subunit in apo form and in Ca2+-bound, NAD-bound, and NADH-bound forms. Structural analyses and comparisons reveal that the αß heterodimer has a similar yet more compact overall structure compared with the αγ heterodimer and contains a pseudo-allosteric site that is structurally different from the allosteric site. In particular, the ß3-α3 and ß12-α8 loops of the ß subunit at the pseudo-allosteric site adopt significantly different conformations from those of the γ subunit at the allosteric site and hence impede the binding of the activators, explaining why the αß heterodimer cannot be allosterically regulated by the activators. The structural data also show that NADH can compete with NAD to bind to the active site and inhibits the activity of the αß heterodimer. These findings together with the biochemical data reveal the molecular basis for the function of the αß heterodimer of human NAD-IDH.

Breaking Cryo-EM Resolution Barriers to Facilitate Drug Discovery.

Recent advances in single-particle cryoelecton microscopy (cryo-EM) are enabling generation of numerous near-atomic resolution structures for well-ordered protein complexes with sizes ≥ ∼200 kDa. Whether cryo-EM methods are equally useful for high-resolution structural analysis of smaller, dynamic protein complexes such as those involved in cellular metabolism remains an important question. Here, we present 3.8 Å resolution cryo-EM structures of the cancer target isocitrate dehydrogenase (93 kDa) and identify the nature of conformational changes induced by binding of the allosteric small-molecule inhibitor ML309. We also report 2.8-Å- and 1.8-Å-resolution structures of lactate dehydrogenase (145 kDa) and glutamate dehydrogenase (334 kDa), respectively. With these results, two perceived barriers in single-particle cryo-EM are overcome: (1) crossing 2 Å resolution and (2) obtaining structures of proteins with sizes < 100 kDa, demonstrating that cryo-EM can be used to investigate a broad spectrum of drug-target interactions and dynamic conformational states.

Crystal structure studies of NADP+ dependent isocitrate dehydrogenase from Thermus thermophilus exhibiting a novel terminal domain.

NADP(+) dependent isocitrate dehydrogenase (IDH) is an enzyme catalyzing oxidative decarboxylation of isocitrate into oxalosuccinate (intermediate) and finally the product α-ketoglutarate. The crystal structure of Thermus thermophilus isocitrate dehydrogenase (TtIDH) ternary complex with citrate and cofactor NADP(+) was determined using X-ray diffraction method to a resolution of 1.80 Å. The overall fold of this protein was resolved into large domain, small domain and a clasp domain. The monomeric structure reveals a novel terminal domain involved in dimerization, very unique and novel domain when compared to other IDH's. And, small domain and clasp domain showing significant differences when compared to other IDH's of the same sub-family. The structure of TtIDH reveals the absence of helix at the clasp domain, which is mainly involved in oligomerization in other IDH's. Also, helices/beta sheets are absent in the small domain, when compared to other IDH's of the same sub family. The overall TtIDH structure exhibits closed conformation with catalytic triad residues, Tyr144-Asp248-Lys191 are conserved. Oligomerization of the protein is quantized using interface area and subunit-subunit interactions between protomers. Overall, the TtIDH structure with novel terminal domain may be categorized as a first structure of subfamily of type IV.

Novel allosteric properties produced by residue substitutions in the subunit interface of yeast NAD+-specific isocitrate dehydrogenase.

Yeast NAD+-specific isocitrate dehydrogenase (IDH) is an octamer of four IDH1 and four IDH2 subunits, and the basic structural unit of the enzyme is an IDH1/IDH2 heterodimer. To investigate one aspect of the interaction between IDH1 and IDH2, residues in a hydrophobic region at the heterodimer interface (Val-216, Ser-220, and Val-224 in IDH1; Ile-221, Val-225, and Val-229 in IDH2) were replaced by alanine residues in each and in both subunits. Gel filtration and sedimentation velocity analyses demonstrated that the residue substitutions do not disrupt the octameric structure of IDH. However, these substitutions produce novel kinetic properties including, with respect to cofactor, positive allosteric regulation by AMP and cooperativity in the absence of AMP. These allosteric properties are also apparent in NAD+-binding experiments. Despite substantial measurable activity for the mutant enzyme containing residue substitutions in both subunits, expression of this enzyme produces growth phenotypes indicative of IDH dysfunction in vivo.