Browsing by Author "Guss, JM"

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    Calmodulin disrupts the structure of the HIV-1 MA protein
    (Elsevier, 2010-07-23) Chow, JYH; Jeffries, CM; Kwan, AH; Guss, JM; Trewhella, J
    The MA protein from HIV-1 is a small, multifunctional protein responsible for regulating various stages of the viral replication cycle. To achieve its diverse tasks, MA interacts with host cell proteins and it has been reported that one of these is the ubiquitous calcium-sensing calmodulin (CaM), which is up-regulated upon HIV-1 infection. The nature of the CaM–MA interaction has been the subject of structural studies, using peptides based on the MA sequence, that have led to conflicting conclusions. The results presented here show that CaM binds intact MA with 1:1 stoichiometry in a Ca2+-dependent manner and that the complex adopts a highly extended conformation in solution as revealed by small-angle X-ray scattering. Alterations in tryptophan fluorescence suggest that the two buried tryptophans (W16 and W36) located in the first two alpha-helices of MA mediate the CaM interaction. Major chemical shift changes occur in the NMR spectrum of MA upon complex formation, whereas chemical shift changes in the CaM spectrum are quite modest and are assigned to residues within the normal target protein-binding hydrophobic clefts of CaM. The NMR data indicate that CaM binds MA via its N- and C-terminal lobes and induces a dramatic conformational change involving a significant loss of secondary and tertiary structure within MA. Circular dichroism experiments suggest that MA loses ~ 20% of its α-helical content upon CaM binding. Thus, CaM binding is expected to impact upon the accessibility of interaction sites within MA that are involved in its various functions. © 2010, Elsevier Ltd.
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    The motif of human cardiac myosin-binding protein C is required for its Ca2+-dependent Interaction with calmoduli
    (American Society for Biochemistry and Molecular Biology, 2012-09-07) Lu, YL; Kwan, AH; Jeffries, CM; Guss, JM; Trewhella, J
    The N-terminal modules of cardiac myosin-binding protein C (cMyBP-C) play a regulatory role in mediating interactions between myosin and actin during heart muscle contraction. The so-called "motif," located between the second and third immunoglobulin modules of the cardiac isoform, is believed to modulate contractility via an "on-off" phosphorylation-dependent tether to myosin Delta S2. Here we report a novel Ca2+-dependent interaction between the motif and calmodulin (CaM) based on the results of a combined fluorescence, NMR, and light and x-ray scattering study. We show that constructs of cMyBP-C containing the motif bind to Ca2+/CaM with a moderate affinity (K-D similar to 10 mu M), which is similar to the affinity previously determined for myosin Delta S2. However, unlike the interaction with myosin Delta S2, the Ca2+/CaM interaction is unaffected by substitution with a triphosphorylated motif mimic. Further, Ca2+/CaM interacts with the highly conserved residues (Glu(319)-Lys(341)) toward the C-terminal end of the motif. Consistent with the Ca2+ dependence, the binding of CaM to the motif is mediated via the hydrophobic clefts within the N- and C-lobes that are known to become more exposed upon Ca2+ binding. Overall, Ca2+/CaM engages with the motif in an extended clamp configuration as opposed to the collapsed binding mode often observed in other CaM-protein interactions. Our results suggest that CaM may act as a structural conduit that links cMyBP-C with Ca2+ signaling pathways to help coordinate phosphorylation events and synchronize the multiple interactions between cMyBP-C, myosin, and actin during the heart muscle contraction. © 2012, American Society for Biochemistry and Molecular Biology.
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    Novel structure of an antikinase and its inhibitor
    (Elsevier, 2011-01-07) Jacques, DA; Langley, DB; Hynson, RMG; Whitten, AE; Kwan, AH; Guss, JM; Trewhella, J
    In Bacillus subtilis, the KipI protein is a regulator of the phosphorelay governing the onset of sporulation. KipI binds the relevant sensor histidine kinase, KinA, and inhibits the autophosphorylation reaction. Gene homologues of kipI are found almost ubiquitously throughout the bacterial kingdom and are usually located adjacent to, and often fused with, kipA gene homologues. In B. subtilis, the KipA protein inhibits the antikinase activity of KipI thereby permitting sporulation. We have used a combination of biophysical techniques in order to understand the domain structure and shape of the KipI–KipA complex and probe the nature of the interaction. We also have solved the crystal structure of TTHA0988, a Thermus thermophilus protein of unknown function that is homologous to a KipI–KipA fusion. This structure, which is the first to be described for this class of proteins, provides unique insight into the nature of the KipI–KipA complex. The structure confirms that KipI and KipA are proteins with two domains, and the C-terminal domains belong to the cyclophilin family. These cyclophilin domains are positioned in the complex such that their conserved surfaces face each other to form a large “bicyclophilin” cleft. We discuss the sequence conservation and possible roles across species of this near-ubiquitous protein family, which is poorly understood in terms of function. © 2011, Elsevier Ltd.
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    Structure of the KinA-Sda complex suggests an allosteric mechanism of histidine kinase inhibition
    (Elsevier, 2007-04-27) Whitten, AE; Jacques, DA; Hammouda, B; Hanley, TL; King, GF; Guss, JM; Trewhella, J; Langley, DB
    The Bacillus subtilis histidine kinase KinA controls activation of the transcription factor governing sporulation, SpoOA. The decision to sporulate involves KinA phosphorylating itself on a conserved histidine residue, after which the phosphate moiety is relayed via two other proteins to SpoOA. The DNA-damage checkpoint inhibitor Sda halts this pathway by binding KinA and blocking the autokinase reaction. We have performed small-angle X-ray scattering and neutron contrast variation studies on the complex formed by KinA and Sda. The data show that two Sda molecules bind to the base of the DHp dimerization domain of the KinA dimer. In this position Sda does riot appear to be able to sterically block the catalytic domain from accessing its target histidine, as previously proposed, but rather may effect an allosteric mode of inhibition involving transmission of the inhibitory signal via the four-helix bundle that forms the DHp domain. © 2007, Elsevier Ltd.
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    Structure of the sporulation histidine kinase inhibitor Sda from bacillus subtilis and insights into its solution state
    (International Union of Crystallography, 2009-06) Jacques, DA; Streamer, M; Rowland, SL; King, GF; Guss, JM; Trewhella, J; Langley, DB
    The crystal structure of the DNA-damage checkpoint inhibitor of sporulation, Sda, from Bacillus subtilis, has been solved by the MAD technique using selenomethionine-substituted protein. The structure closely resembles that previously solved by NMR, as well as the structure of a homologue from Geobacillus stearothermophilus solved in complex with the histidine kinase KinB. The structure contains three molecules in the asymmetric unit. The unusual trimeric arrangement, which lacks simple internal symmetry, appears to be preserved in solution based on an essentially ideal fit to previously acquired scattering data for Sda in solution. This interpretation contradicts previous findings that Sda was monomeric or dimeric in solution. This study demonstrates the difficulties that can be associated with the characterization of small proteins and the value of combining multiple biophysical techniques. It also emphasizes the importance of understanding the physical principles behind these techniques and therefore their limitations. © 2009, International Union of Crystallography
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    The structure of TTHA0988 from thermus thermophilus, a KipI-KipA homologue incorrectly annotated as an allophanate hydrolase
    (Wiley-Blackwell, 2011-02-01) Jacques, DA; Langley, DB; Kuramitsu, S; Yokoyama, S; Trewhella, J; Guss, JM
    The Thermus thermophilus protein TTHA0988 is a protein of unknown function which represents a fusion of two proteins found almost ubiquitously across the bacterial kingdom. These two proteins perform a role regulating sporulation in Bacillus subtilis, where they are known as KipI and KipA. kipI and kipA genes are usually found immediately adjacent to each other and are often fused to produce a single polypeptide, as is the case with TTHA0988. Here, three crystal forms are reported of TTHA0988, the first structure to be solved from the family of `KipI-KipA fusion' proteins. Comparison of the three forms reveals structural flexibility which can be described as a hinge motion between the `KipI' and `KipA' components. TTHA0988 is annotated in various databases as a putative allophanate hydrolase. However, no such activity could be identified and genetic analysis across species with known allophanate hydrolases indicates that a misannotation has occurred. © 2011, Wiley-Blackwell. The definitive version is available at www3.interscience.wiley.com