Browsing by Author "Langley, DB"

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    Ligand-induced conformational changes and conformational dynamics in the solution structure of the lactose repressor protein
    (Elsevier, 2008-02-15) Taraban, M; Zhan, HL; Whitten, AE; Langley, DB; Matthews, KS; Swint-Kruse, L; Trewhella, J
    We present here the results of a series of small-angle X-ray scattering studies aimed at understanding the role of conformational changes and structural flexibility in DNA binding and allosteric signaling in a bacterial transcription regulator, lactose repressor protein (LacI). Experiments were designed to detect possible conformational changes that occur when LacI binds either DNA or the inducer IPTG, or both. Our studies included the native LacI dimer of homodimers and a dimeric variant (R3), enabling us to probe conformational changes within the homodimers and distinguish them from those involving changes in the homodimer-homodimer relationships. The scattering data indicate that removal of operator DNA (oDNA) from R3 results in an unfolding and extension of the hinge helix that connects the LacI regulatory and DNA-binding domains. In contrast, only very subtle conformational changes occur in the R3 dimer-oDNA complex upon IPTG binding, indicative of small adjustments in the orientations of domains and/or subdomains within the structure. The binding of IPTG to native (tetrameric) LacI-oDNA complexes also appears to facilitate a modest change in the average homodimer-homodimer disposition. Notably, the crystal structure of the native LacI-oDNA complex differs significantly from the average solution conformation. The solution scattering data are best fit by an ensemble of structures that includes (1) similar to 60% of the V-shaped dimer of homodimers observed in the crystal structure and (2) similar to 40% of molecules with more "open" forms, such as those generated when the homodimers move with respect to each other about the tetramerization domain: In gene regulation, such a flexible LacI would be beneficial for the interaction of its two DNA-binding domains, positioned at the tips of the V, with the required two of three LacI operators needed for full repression. © 2007, Elsevier Ltd.
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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