Browsing by Author "Kwan, AH"
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- ItemCalmodulin binds a highly extended HIV-1 MA protein that refolds upon its release(Cell Press, 2012-08-08) Taylor, JE; Chow, JYH; Jeffries, CM; Kwan, AH; Duff, AP; Hamilton, WA; Trewhella, JCalmodulin (CaM) expression is upregulated upon HIV-1 infection and interacts with proteins involved in viral processing, including the multifunctional HIV-1 MA protein. We present here the results of studies utilizing small-angle neutron scattering with contrast variation that, when considered in the light of earlier fluorescence and NMR data, show CaM binds MA in an extended open-clamp conformation via interactions with two tryptophans that are widely spaced in sequence and space. The interaction requires a disruption of the MA tertiary fold such that MA becomes highly extended in a long snakelike conformation. The CaM-MA interface is extensive, covering ∼70% of the length of the MA such that regions known to be important in MA interactions with critical binding partners would be impacted. The CaM conformation is semiextended and as such is distinct from the classical CaM-collapse about short α-helical targets. NMR data show that upon dissociation of the CaM-MA complex, either by the removal of Ca2+ or increasing ionic strength, MA reforms its native tertiary contacts. Thus, we observe a high level of structural plasticity in MA that may facilitate regulation of its activities via intracellular Ca2+-signaling during viral processing. © 2012 Biophysical Society.
- ItemCalmodulin disrupts the structure of the HIV-1 MA protein(Elsevier, 2010-07-23) Chow, JYH; Jeffries, CM; Kwan, AH; Guss, JM; Trewhella, JThe 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.
- ItemThe 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, JThe 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.
- ItemNovel structure of an antikinase and its inhibitor(Elsevier, 2011-01-07) Jacques, DA; Langley, DB; Hynson, RMG; Whitten, AE; Kwan, AH; Guss, JM; Trewhella, JIn 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.
- ItemSolid-state NMR spectroscopy of functional amyloid from a fungal hydrophobin: a well-ordered β-sheet core amidst structural heterogeneity(Wiley-VCH Verlag GMBH, 2012-01-01) Morris, VK; Linser, R; Wilde, KL; Duff, AP; Sunde, M; Kwan, AHGrEASy fibrils: Hydrophobins are fungal proteins that assemble into an amphipathic fibrillar monolayer with amyloid properties and a hydrophobic face as water-resistant as Teflon. Solid-state NMR studies on EAS hydrophobin fibrils reveal direct evidence of a partial molecular rearrangement on assembly and an ordered β-sheet-rich core in the context of a whole protein in this functional amyloid. © 2012, Wiley-VCH Verlag GmbH & Co. KGaA
- ItemSolution structure of the LIM-homeodomain transcription factor complex Lhx3/Ldb1 and the effects of a pituitary mutation on key Lhx3 interactions(Public Libary of Science, 2012-07-25) Bhati, M; Lee, C; Gadd, MS; Jeffries, CM; Kwan, AH; Whitten, AE; Trewhella, J; Mackay, JP; Matthews, JMLhx3 is a LIM-homeodomain (LIM-HD) transcription factor that regulates neural cell subtype specification and pituitary development in vertebrates, and mutations in this protein cause combined pituitary hormone deficiency syndrome (CPHDS). The recently published structures of Lhx3 in complex with each of two key protein partners, Isl1 and Ldb1, provide an opportunity to understand the effect of mutations and posttranslational modifications on key protein-protein interactions. Here, we use small-angle X-ray scattering of an Ldb1-Lhx3 complex to confirm that in solution the protein is well represented by our previously determined NMR structure as an ensemble of conformers each comprising two well-defined halves (each made up of LIM domain from Lhx3 and the corresponding binding motif in Ldb1) with some flexibility between the two halves. NMR analysis of an Lhx3 mutant that causes CPHDS, Lhx3(Y114C), shows that the mutation does not alter the zinc-ligation properties of Lhx3, but appears to cause a structural rearrangement of the hydrophobic core of the LIM2 domain of Lhx3 that destabilises the domain and/or reduces the affinity of Lhx3 for both Ldb1 and Isl1. Thus the mutation would affect the formation of Lhx3-containing transcription factor complexes, particularly in the pituitary gland where these complexes are required for the production of multiple pituitary cell types and hormones. © 2012 Bhati et al.