Jimin Zheng

Structure of the bifunctional isocitrate dehydrogenase kinase/phosphatase.

The Escherichia coli isocitrate dehydrogenase kinase/phosphatase (AceK) is a unique bifunctional enzyme that phosphorylates or dephosphorylates isocitrate dehydrogenase (ICDH) in response to environmental changes, resulting in the inactivation or, respectively, activation of ICDH. ICDH inactivation short-circuits the Krebs cycle by enabling the glyoxlate bypass. It was the discovery of AceK and ICDH that established the existence of protein phosphorylation regulation in prokaryotes. As a 65-kDa protein, AceK is significantly larger than typical eukaryotic protein kinases. Apart from the ATP-binding motif, AceK does not share sequence homology with any eukaryotic protein kinase or phosphatase. Most intriguingly, AceK possesses the two opposing activities of protein kinase and phosphatase within one protein, and specifically recognizes only intact ICDH. Additionally, AceK has strong ATPase activity. It has been shown that AceK kinase, phosphatase and ATPase activities reside at the same site, although the molecular basis of such multifunctionality and its regulation remains completely unknown. Here we report the structures of AceK and its complex with ICDH. The AceK structure reveals a eukaryotic protein-kinase-like domain containing ATP and a regulatory domain with a novel fold. As an AceK phosphatase activator and kinase inhibitor, AMP is found to bind in an allosteric site between the two AceK domains. An AMP-mediated conformational change exposes and shields ATP, acting as a switch between AceK kinase and phosphatase activities, and ICDH-binding induces further conformational change for AceK activation. The substrate recognition loop of AceK binds to the ICDH dimer, allowing higher-order substrate recognition and interaction, and inducing critical conformational change at the phosphorylation site of ICDH.

The complex structure of calmodulin bound to a calcineurin peptide.

The activity of the protein phosphatase calcineurin (CN) is regulated by an autoinhibition mechanism wherein several domains from its catalytic A subunit, including the calmodulin binding domain (CaMBD), block access to its active site. Upon binding of Ca2+ and calmodulin (Ca2+/CaM) to CaMBD, the autoinhibitory domains dissociate from the catalytic groove, thus activating the enzyme. To date, the structure of the CN/CaM/Ca2+ complex has not been determined in its entirety. Previously, we determined the structure of a fusion protein consisting of CaM and a 25‐residue peptide taken from the CaMBD, joined by a 5‐glycine linker. This structure revealed a novel CaM binding motif. However, the presence of the extraneous glycine linker cast doubt on the authenticity of this structure as an accurate representation of CN/CaM binding in vivo. Thus, here, we have determined the crystal structure of CaM complexed with the 25‐residue CaMBD peptide without the glycine linker at a resolution of 2.1 Å. The structure is essentially identical to the fusion construction which displays CaM bound to the CaMBD peptide as a dimer with an open, elongated conformation. The N‐lobe from one molecule and C‐lobe from another encompass and bind the CaMBD peptide. Thus, it validates the existence of this novel CaM binding motif. Our experiments suggest that the dimeric CaM/CaMBD complex exists in solution, which is unambiguously validated using a carefully‐designed CaM‐sepharose pull‐down experiment. We discuss structural features that produce this novel binding motif, including the role of the CaMBD peptide residues Arg‐408, Val‐409, and Phe‐410, which work to provide rigidity to the otherwise flexible central CaM helix joining the N‐ and C‐lobes, ultimately keeping these lobes apart and forcing “head‐to‐tail” dimerization to attain the requisite N‐ and C‐lobe pairing for CaMBD binding. Proteins 2008.

Crystal structure of a novel prokaryotic Ser/Thr kinase and its implication in the Cpx stress response pathway

The Cpx signalling system of Escherichia coli and Salmonella enterica senses extracytoplasmic stress and controls expression of factors that allow the bacterium to adapt to these stressors and thereby enhance survival. Many of the Cpx‐responsive genes products are of unknown function. We determined the crystal structure of one of these gene products, called YihE in E. coli, which exhibits a eukaryotic kinase fold. Functional assays established that both YihE and the S. enterica YihE homologue, RdoA, undergo autophosphorylation and phosphorylate protein substrates at Ser/Thr residues in vitro, demonstrating that YihE/RdoA is a novel Ser/Thr protein kinase in prokaryotic cells. Phenotypic analysis of yihE/rdoA null strains indicates that this kinase is most abundant in stationary phase, and is important for long‐term cell survival and for expression of surface appendages in both a Cpx‐independent and ‐dependent manner. YihE/RdoA is therefore a previously unknown kinase component of a new type of bacterial phosphorelay mechanism, adding kinase activity as another response to the Cpx sensing system that functions to maintain cellular homeostasis.

Contributions of F-BAR and SH2 Domains of Fes Protein Tyrosine Kinase for Coupling to the FcεRI Pathway in Mast Cells

ABSTRACT This study investigates the roles of Fer-CIP4 homology (FCH)-Bin/amphiphysin/Rvs (F-BAR) and SH2 domains of Fes protein tyrosine kinase in regulating its activation and signaling downstream of the high-affinity immunoglobulin G (IgE) receptor (FcεRI) in mast cells. Homology modeling of the Fes F-BAR domain revealed conservation of some basic residues implicated in phosphoinositide binding (R113/K114). The Fes F-BAR can bind phosphoinositides and induce tubulation of liposomes in vitro. Mutation of R113/K114 to uncharged residues (RK/QQ) caused a significant reduction in phosphoinositide binding in vitro and a more diffuse cytoplasmic localization in transfected COS-7 cells. RBL-2H3 mast cells expressing full-length Fes carrying the RK/QQ mutation show defects in FcεRI-induced Fes tyrosine phosphorylation and degranulation compared to cells expressing wild-type Fes. This correlated with reduced localization to Lyn kinase-containing membrane fractions for the RK/QQ mutant compared to wild-type Fes in mast cells. The Fes SH2 domain also contributes to Fes signaling in mast cells, via interactions with the phosphorylated FcεRI β chain and the actin regulatory protein HS1. We show that Fes phosphorylates C-terminal tyrosine residues in HS1 implicated in actin stabilization. Thus, coordinated actions of the F-BAR and SH2 domains of Fes allow for coupling to FcεRI signaling and potential regulation the actin reorganization in mast cells.

Structural and mechanistic insights into the bifunctional enzyme isocitrate dehydrogenase kinase/phosphatase AceK.

The switch between the Krebs cycle and the glyoxylate bypass is controlled by isocitrate dehydrogenase kinase/phosphatase (AceK). AceK, a bifunctional enzyme, phosphorylates and dephosphorylates isocitrate dehydrogenase (IDH) with its unique active site that harbours both the kinase and ATP/ADP-dependent phosphatase activities. AceK was the first example of prokaryotic phosphorylation identified, and the recent characterization of the structures of AceK and its complex with its protein substrate, IDH, now offers a new understanding of both previous and future endeavours. AceK is structurally similar to the eukaryotic protein kinase superfamily, sharing many of the familiar catalytic and regulatory motifs, demonstrating a close evolutionary relationship. Although the active site is shared by both the kinase and phosphatase functions, the catalytic residues needed for phosphatase function are readily seen when compared with the DXDX(T/V) family of phosphatases, despite the fact that the phosphatase function of AceK is strictly ATP/ADP-dependent. Structural analysis has also allowed a detailed look at regulation and its stringent requirements for interacting with IDH.

Structural basis of the substrate specificity of bifunctional isocitrate dehydrogenase kinase/phosphatase

Isocitrate dehydrogenase kinase/phosphatase (AceK) regulates entry into the glyoxylate bypass by reversibly phosphorylating isocitrate dehydrogenase (ICDH). On the basis of the recently determined structure of the AceK-ICDH complex from Escherichia coli, we have classified the structures of homodimeric NADP(+)-ICDHs to rationalize and predict which organisms likely contain substrates for AceK. One example is Burkholderia pseudomallei (Bp). Here we report a crystal structure of Bp-ICDH that exhibits the necessary structural elements required for AceK recognition. Kinetic analyses provided further confirmation that Bp-ICDH is a substrate for AceK. We conclude that the highly stringent AceK binding sites on ICDH are maintained only in Gram-negative bacteria.

Expression and preliminary characterization of human MICU2

ABSTRACT MICU2 has been reported to interact with MICU1 and participate in the regulation of mitochondrial Ca2+ uptake, although the molecular determinants underlying the function of MICU2 is unknown. In order to characterize MICU2 we screened a series of N-terminal and C-terminal truncations and obtained constructs which can be expressed in abundance, giving rise to soluble samples to enable subsequent characterizations. Size exclusion chromatography (SEC) and multi-angle laser light scattering (MALLS) revealed that MICU2 exists as a monomer in Ca2+-free conditions but forms a dimer in Ca2+-bound conditions. Unlike MICU1, the C-helix domain of MICU2 exhibits no influence on protein conformation in both Ca2+-free and Ca2+-bound forms. Furthermore, mutation of the first EF-hand abolishes the ability of MICU2 to switch to a dimer in the presence of Ca2+, indicating that the first EF-hand is not only involved in Ca2+ binding but also in conformational change. Our pull-down and co-immunoprecipitation assays suggest that, in addition to disulfide bonds, salt bridges also contribute to MICU1-MICU2 heterodimer formation. Summary: Ca2+ induces a monomer-dimer switch of MICU2, with the associated conformational change governed by its first EF-hand, but not influenced by the C-helix domain. A disulfide bond and salt bridge each contribute to MICU1-MICU2 heterodimer formation.

Insights into the Cellular Function of YhdE, a Nucleotide Pyrophosphatase from Escherichia coli

YhdE, a Maf-like protein in Escherichia coli, exhibits nucleotide pyrophosphatase (PPase) activity, yet its cellular function remains unknown. Here, we characterized the PPase activity of YhdE on dTTP, UTP and TTP and determined two crystal structures of YhdE, revealing ‘closed’ and ‘open’ conformations of an adaptive active site. Our functional studies demonstrated that YhdE retards cell growth by prolonging the lag and log phases, particularly under stress conditions. Morphology studies showed that yhdE-knockout cells transformed the normal rod shape of wild-type cells to a more spherical form, and the cell wall appeared to become more flexible. In contrast, YhdE overexpression resulted in filamentous cells. This study reveals the previously unknown involvement of YhdE in cell growth inhibition under stress conditions, cell-division arrest and cell-shape maintenance, highlighting YhdE’s important role in E. coli cell-cycle checkpoints.

Structures of an Eph receptor tyrosine kinase and its potential activation mechanism.

Eph receptor tyrosine kinases (RTKs) and their ephrin ligands play a crucial role in both physiological and pathophysiological processes, including tumourigenesis. A previous study of Eph RTKs established a regulatory role for the juxtamembrane segment (JMS) in kinase activation through the phosphorylation of two tyrosines within the JMS. Here, structures of EphA2 representing various activation states are presented. By determining the unphosphorylated inactive and phosphorylated active structures as well as an alternative conformation, conformational changes during kinase activation have been revealed. It is shown that phosphorylation of a tyrosine residue (Tyr772) in the activation loop without direct involvement of the JMS is sufficient to activate the EphA2 kinase. This mechanistic finding is in contrast to the mechanism of other Eph RTKs, such as EphB2, in which phosphorylation of the two JMS tyrosines initiates the dissociation of the JMS and triggers activation-loop phosphorylation for kinase activation. Furthermore, experiments demonstrate that the EphA2 substrate PTEN, a phosphatase that has been implicated in tumour suppression, acts to regulate the phosphorylation states of EphA2, exemplifying a unique reciprocal enzyme-substrate system. Based on these studies, it is therefore suggested that EphA2 may possess an alternate activation mechanism distinct from other Eph RTKs.

Unique Kinase Catalytic Mechanism of AceK with a Single Magnesium Ion

Isocitrate dehydrogenase kinase/phosphatase (AceK) is the founding member of the protein phosphorylation system in prokaryotes. Based on the novel and unique structural characteristics of AceK recently uncovered, we sought to understand its kinase reaction mechanism, along with other features involved in the phosphotransfer process. Herein we report density functional theory QM calculations of the mechanism of the phosphotransfer reaction catalysed by AceK. The transition states located by the QM calculations indicate that the phosphorylation reaction, catalysed by AceK, follows a dissociative mechanism with Asp457 serving as the catalytic base to accept the proton delivered by the substrate. Our results also revealed that AceK prefers a single Mg2+-containing active site in the phosphotransfer reaction. The catalytic roles of conserved residues in the active site are discussed.