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Structural basis of histidine kinase autophosphorylation deduced by integrating genomics, molecular dynamics, and mutagenesis.

ABSTRACT: Signal transduction proteins such as bacterial sensor histidine kinases, designed to transition between multiple conformations, are often ruled by unstable transient interactions making structural characterization of all functional states difficult. This study explored the inactive and signal-activated conformational states of the two catalytic domains of sensor histidine kinases, HisKA and HATPase. Direct coupling analyses, a global statistical inference approach, was applied to >13,000 such domains from protein databases to identify residue contacts between the two domains. These contacts guided structural assembly of the domains using MAGMA, an advanced molecular dynamics docking method. The active conformation structure generated by MAGMA simultaneously accommodated the sequence derived residue contacts and the ATP-catalytic histidine contact. The validity of this structure was confirmed biologically by mutation of contact positions in the Bacillus subtilis sensor histidine kinase KinA and by restoration of activity in an inactive KinA(HisKA):KinD(HATPase) hybrid protein. These data indicate that signals binding to sensor domains activate sensor histidine kinases by causing localized strain and unwinding at the end of the C-terminal helix of the HisKA domain. This destabilizes the contact positions of the inactive conformation of the two domains, identified by previous crystal structure analyses and by the sequence analysis described here, inducing the formation of the active conformation. This study reveals that structures of unstable transient complexes of interacting proteins and of protein domains are accessible by applying this combination of cross-validating technologies.

SUBMITTER: Dago AE

PROVIDER: S-EPMC3387055 | biostudies-literature | 2012 Jun

REPOSITORIES: biostudies-literature

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Structural basis of histidine kinase autophosphorylation deduced by integrating genomics, molecular dynamics, and mutagenesis.

Dago Angel E AE Schug Alexander A Procaccini Andrea A Hoch James A JA Weigt Martin M Szurmant Hendrik H

Proceedings of the National Academy of Sciences of the United States of America 20120605 26

Signal transduction proteins such as bacterial sensor histidine kinases, designed to transition between multiple conformations, are often ruled by unstable transient interactions making structural characterization of all functional states difficult. This study explored the inactive and signal-activated conformational states of the two catalytic domains of sensor histidine kinases, HisKA and HATPase. Direct coupling analyses, a global statistical inference approach, was applied to >13,000 such do ...[more]

PMID: 22670053

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Project description:BackgroundProtein Kinase Casein Kinase 2 (PKCK2) is an ubiquitous Ser/Thr kinase expressed in all eukaryotes. It phosphorylates a number of proteins involved in various cellular processes. PKCK2 holoenzyme is catalytically active tetramer, composed of two homologous or identical and constitutively active catalytic (alpha) and two identical regulatory (beta) subunits. The tetramer cannot phosphorylate some substrates that can be phosphorylated by PKCK2alpha in isolation. The present work explores the structural basis of this feature using computational analysis and modeling.ResultsWe have initially built a model of PKCK2alpha bound to a substrate peptide with a conformation identical to that of the substrates in the available crystal structures of other kinases complexed with the substrates/ pseudosubstrates. In this model however, the fourth acidic residue in the consensus pattern of the substrate, S/T-X-X-D/E where S/T is the phosphorylation site, did not result in interaction with the active form of PKCK2alpha and is highly solvent exposed. Interaction of the acidic residue is observed if the substrate peptide adopts conformations as seen in beta turn, alpha helix, or 3(10) helices. This type of conformation is observed and accommodated well by PKCK2alpha in calmodulin where the phosphorylation site is at the central helix. PP2A carries sequence patterns for PKCK2alpha phosphorylation. While the possibility of PP2A being phosphorylated by PKCK2 has been raised in the literature we use the model of PP2A to generate a model of PP2A-PKCK2alpha complex. PKCK2beta undergoes phosphorylation by holoenzyme at the N-terminal region, and is accommodated very well in the limited space available at the substrate-binding site of the holoenzyme while the space is insufficient to accommodate the binding of PP2A or calmodulin in the holoenzyme.ConclusionCharge and shape complimentarity seems to play a role in substrate recognition and binding to PKCK2alpha, along with the consensus pattern. The detailed conformation of the substrate peptide binding to PKCK2 differs from the conformation of the substrate/pseudo substrate peptide that is bound to other kinases in the crystal structures reported. The ability of holoenzyme to phosphorylate substrate proteins seems to depend on the accessibility of the P-site in limited space available in holoenzyme.

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Structural basis of histidine kinase autophosphorylation deduced by integrating genomics, molecular dynamics, and mutagenesis.

Publications

Structural basis of histidine kinase autophosphorylation deduced by integrating genomics, molecular dynamics, and mutagenesis.

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