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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.

Section: Methodsmentioning

confidence: 99%

Structural and Enzymatic Insights into the ATP Binding and Autophosphorylation Mechanism of a Sensor Histidine Kinase

Trajtenberg¹,

Graña

Ruétalo³

et al. 2010

“…This structural homology is remarkable given that only the ADP/ATP moiety was used to fit these molecules. In addition, x-ray structural studies have shown that there is extremely high structural homology within the ATP-binding pocket in the histidine kinases and ATPases (29).…”

Section: Fig 2 Alignment Of the Pdk2 Nucleotide-binding Domainmentioning

confidence: 99%

Structure of Pyruvate Dehydrogenase Kinase

Steussy

Popov

Bowker-Kinley

et al. 2001

The structure of mitochondrial pyruvate dehydrogenase kinase isozyme 2 is of interest because it represents a family of serine-specific protein kinases that lack sequence similarity with all other eukaryotic protein kinases. Similarity exists instead with key motifs of prokaryotic histidine protein kinases and a family of eukaryotic ATPases. The 2.5-Å crystal structure reported here reveals that pyruvate dehydrogenase kinase isozyme 2 has two domains of about the same size. The N-terminal half is dominated by a bundle of four amphipathic ␣-helices, whereas the C-terminal half is folded into an ␣/␤ sandwich that contains the nucleotide-binding site. Analysis of the structure reveals this C-terminal domain to be very similar to the nucleotidebinding domain of bacterial histidine kinases, but the catalytic mechanism appears similar to that of the eukaryotic serine kinases and ATPases.

“…All necessary elements for histidine phosphorylation are found in the phosphotransfer (P1) and kinase (P4) domains of the five-domain CheA protein (4). Conserved residues in the P4 domain bind ATP in a pocket that optimally positions the ␥-phosphoryl for transfer to a specific histidine on the N-terminal P1 domain (4,5).…”

mentioning

confidence: 99%

Structural and Chemical Requirements for Histidine Phosphorylation by the Chemotaxis Kinase CheA

Quezada

Hamel

Grădinaru

et al. 2005

Self Cite

The CheA histidine kinase initiates the signal transduction pathway of bacterial chemotaxis by autophosphorylating a conserved histidine on its phosphotransferase domain (P1). Site-directed mutations of neighboring conserved P1 residues (Glu-67, Lys-48, and His-64) show that a hydrogen-bonding network controls the reactivity of the phospho-accepting His (His-45) in Thermotoga maritima CheA. In particular, the conservative mutation E67Q dramatically reduces phosphotransfer to P1 without significantly affecting the affinity of P1 for the CheA ATP-binding domain. High resolution crystallographic studies revealed that although all mutants disrupt the hydrogen-bonding network to varying degrees, none affect the conformation of His-45.15 N-NMR chemical shift studies instead showed that Glu-67 functions to stabilize the unfavored N ␦1 H tautomer of His-45, thereby rendering the N ⑀2 imidazole unprotonated and well positioned for accepting the ATP phosphoryl group.