Abstract
DesK is a sensor histidine kinase (HK) that allows Bacillus subtilis to respond to cold shock, triggering the adaptation of membrane fluidity via transcriptional control of a fatty acid desaturase. It belongs to the HK family HPK7, which includes the nitrogen metabolism regulators NarX/Q and the antibiotic sensor LiaS among other important sensor kinases. Structural information on different HK families is still scarce and several questions remain, particularly concerning the molecular features that determine HK specificity during its catalytic autophosphorylation and subsequent response-regulator phosphotransfer reactions. To analyze the ATP-binding features of HPK7 HKs and dissect their mechanism of autophosphorylation at the molecular level, we have studied DesK in complex with ATP using high resolution structural approaches in combination with biochemical studies. We report the first crystal structure of an HK in complex with its natural nucleotidic substrate. The general fold of the ATP-binding domain of DesK is conserved, compared with well studied members of other families. Yet, DesK displays a far more compact structure at the ATP-binding pocket: the ATP lid loop is much shorter with no secondary structural organization and becomes ordered upon ATP loading. Sequence conservation mapping onto the molecular surface, semi-flexible protein-protein docking simulations, and structure-based point mutagenesis allow us to propose a specific domain-domain geometry during autophosphorylation catalysis. Supporting our hypotheses, we have been able to trap an autophosphorylating intermediate state, by protein engineering at the predicted domain-domain interaction surface.
Highlights
Intracellular histidine kinase (HK) domains catalyze an ATP-dependent protein kinase activity, allowing the Histidine kinases3 (HKs) to autophosphorylate a specific histidine, in a signal-regulated fashion
ATP Binding and Autophosphorylation in Histidine Kinases transfer domain immediately precedes the ATP-binding domain (ABD) and mediates HK dimerization ( called the dimerization and histidine phosphotransfer (DHp) domain), whereas in class II enzymes it is not involved in dimerization and several additional domains intervene between the phosphotransfer and ABD domains
According to the DHp domains, HKs have been grouped into four Pfam families [9]: whereas ϳ85% can be assigned to the HisKA group (PF00512), ϳ10% of all known histidine kinases belong to the HisKA_3 family (PF007730)
Summary
Transfer domain immediately precedes the ABD and mediates HK dimerization ( called the dimerization and histidine phosphotransfer (DHp) domain), whereas in class II enzymes it is not involved in dimerization and several additional domains intervene between the phosphotransfer and ABD domains. In vivo experiments have shown that DesK functions as a kinase at cold temperatures [15, 16], autophosphorylating a conserved residue (His188) in the DHp domain. This phosphoryl group is transferred to a receiver aspartate in DesR, a DNA-binding RR that activates the transcription of des (encoding for the acyl lipid desaturase ⌬5-Des). Structural analyses, complemented by an extensive in silico study of sequence conservation and phosphotransfer intermediate-state modeling, allow us to extend the current knowledge of HKs. In particular, we pinpoint key residues at the domain-domain interaction surface, enabling us to predict the geometry of the interdomain interface during autophosphorylation. Further supporting the structural hypotheses, we engineered cysteine mutants on the basis of the predicted interdomain orientation, allowing us to trap a covalent autophosphorylation intermediate state
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