Intersubunit Ionic Interactions Stabilize the Nucleoside Diphosphate Kinase of Mycobacterium tuberculosis

Abstract

Most nucleoside diphosphate kinases (NDPKs) are hexamers. The C-terminal tail interacting with the neighboring subunits is crucial for hexamer stability. In the NDPK from Mycobacterium tuberculosis (Mt) this tail is missing. The quaternary structure of Mt-NDPK is essential for full enzymatic activity and for protein stability to thermal and chemical denaturation. We identified the intersubunit salt bridge Arg80-Asp93 as essential for hexamer stability, compensating for the decreased intersubunit contact area. Breaking the salt bridge by the mutation D93N dramatically decreased protein thermal stability. The mutation also decreased stability to denaturation by urea and guanidinium. The D93N mutant was still hexameric and retained full activity. When exposed to low concentrations of urea it dissociated into folded monomers followed by unfolding while dissociation and unfolding of the wild type simultaneously occur at higher urea concentrations. The dissociation step was not observed in guanidine hydrochloride, suggesting that low concentration of salt may stabilize the hexamer. Indeed, guanidinium and many other salts stabilized the hexamer with a half maximum effect of about 0.1 M, increasing protein thermostability. The crystal structure of the D93N mutant has been solved.

Hydrogen/Deuterium Exchange Mass Spectrometry Reveals Mechanistic Details of Activation of Nucleoside Diphosphate Kinases by Oligomerization

“…(57) We recently showed in vitro that isolated sub-hexameric assemblies of Mt-NDPK are inactive, complete assembly being essential for function. (53) By combining crystallography and biochemistry, we also demonstrated that hexamer's high thermostability (76°C) is dependent on six inter-subunit R80-D93 salt bridges. (54) The point mutation D93N reduces the melting point by 28°C, without significant alteration of the hexameric 3D structure or enzymatic properties.…”

“…(54) The point mutation D93N reduces the melting point by 28°C, without significant alteration of the hexameric 3D structure or enzymatic properties. (53) hexamers and dimers explain why hexamerization is required for kinase activity.…”

“…They are in full agreement with our previous data on Mt-NDPK as well as with other similar studies on NDPK which show hysteresis after about 1 day of unfolding/refolding in presence of denaturing agents. (42,53) Having established that unfolding and refolding pathways were different for both WT and D93N…”

“…In addition, the stabilization of hexamers by subunit interactions has been studied. (49)(50)(51)(52)(53) Crystallographic structures of several-NDPK mutants provide detailed understanding of the WT stabilization mechanism, (53,54) but provide little understanding as to why oligomerization is necessary for function. So, we realized that by combining stability aspects with the study of their dynamic properties, we would improve this understanding.…”

Nucleoside diphosphate kinases (NDPKs) in animal development

“…One unifying idea from Lascu and colleagues is that the S120G mutation affects the quaternary structure of the NDPK hexamer; e.g., it generates an unstable molten globule form of NDPK that cannot form the highly active hexamer [114,115].…”