Browsing by Author "Jeffries, CM"

Now showing 1 - 17 of 17
  • Thumbnail Image
    Item
    Calmodulin binds a highly extended HIV-1 MA protein that refolds upon its release
    (Cell Press, 2012-08-08) Taylor, JEN; Chow, JYH; Jeffries, CM; Kwan, AH; Duff, AP; Hamilton, WA; Trewhella, J
    Calmodulin (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.
  • No Thumbnail Available
    Item
    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.
  • No Thumbnail Available
    Item
    Cardiac myosin-binding protein C decorates F-actin: implications for cardiac function
    (National Academy of Sciences, 2008-11-25) Whitten, AE; Jeffries, CM; Harris, SP; Trewhella, J
    Cardiac myosin-binding protein C (cMyBP-C) is an accessory protein of striated muscle sarcomeres that is vital for maintaining regular heart function. Its 4 N-terminal regulatory domains, C0-C1-m-C2 (C0C2), influence actin and myosin interactions, the basic contractile proteins of muscle. Using neutron contrast variation data, we have determined that C0C2 forms a repeating assembly with filamentous actin, where the C0 and C1 domains of C0C2 attach near the DNase I-binding loop and subdomain 1 of adjacent actin monomers. Direct interactions between the N terminus of cMyBP-C and actin thereby provide a mechanism to modulate the contractile cycle by affecting the regulatory state of the thin filament and its ability to interact with myosin. © 2008, National Academy of Sciences
  • Thumbnail Image
    Item
    (Corrected) Calmodulin binds a highly extended HIV-1 MA protein that refolds upon its release
    (Elsevier, 2023-05-02) Taylor, JEN; Chow, JYH; Jeffries, CM; Kwan, AH; Duff, AP; Hamilton, WA; Trewhella, J
    (Biophysical Journal 103, August 2012; 541–549) The authors identified an omission in this article. The figure legend for Figure S2 should include the following: “The model displayed here includes full-length calcium-bound CaM and the matrix protein sequence spanning amino acids 1–113; i.e., it does not include the flexible C-terminal tail (amino acids 114–133).” None of the article's conclusions are affected by this omission. In addition, after publication of the article, the SAXS and SANS data and modeling for this article were deposited in the Small Angle Scattering Biological Data Bank (SASBDB; https://www.sasbdb.org/) under accession code SASDKR3. Finally, the first author's name should appear as “James E. N. Taylor” rather than “James E. Taylor.”
  • No Thumbnail Available
    Item
    Effects of macromolecular crowding on an intrinsically disordered protein characterized by small-angle neutron scattering with contrast matching
    (Elsevier (Cell Press), 2011-02-16) Johansen, D; Jeffries, CM; Hammouda, B; Trewhella, J; Goldenberg, DP
    Small-angle neutron scattering was used to examine the effects of molecular crowding on an intrinsically disordered protein, the N protein of bacteriophage lambda, in the presence of high concentrations of a small globular protein, bovine pancreatic trypsin inhibitor (BPTI). The N protein was labeled with deuterium, and the D2O concentration of the solvent was adjusted to eliminate the scattering contrast between the solvent and unlabeled BPTI, leaving only the scattering signal from the unfolded protein. The scattering profile observed in the absence of BPTI closely matched that predicted for an ensemble of random conformations. With BPTI added to a concentration of 65 mg/mL, there was a clear change in the scattering profile representing an increase in the mass fractal dimension of the unfolded protein, from 1.7 to 1.9, as expected if crowding favors more compact conformations. The crowding protein also inhibited aggregation of the unfolded protein. At 130 mg/mL BPTI, however, the fractal dimension was not significantly different from that measured at the lower concentration, contrary to the predictions of models that treat the unfolded conformations as convex particles. These results are reminiscent of the behavior of polymers in concentrated melts, suggesting that these synthetic mixtures may provide useful insights into the properties of unfolded proteins under crowding conditions. © 2011, Cell Press
  • No Thumbnail Available
    Item
    Invited review: probing the structures of muscle regulatory proteins using small-angle solution scattering
    (Wiley-Blackwell, 2011-08-01) Lu, YL; Jeffries, CM; Trewhella, J
    Small-angle X-ray and neutron scattering with contrast variation have made important contributions in advancing our understanding of muscle regulatory protein structures in the context of the dynamic molecular processes governing muscle action. The contributions of the scattering investigations have depended upon the results of key crystallographic, NMR, and electron microscopy experiments that have provided detailed structural information that has aided in the interpretation of the scattering data. This review will cover the advances made using small-angle scattering techniques, in combination with the results from these complementary techniques, in probing the structures of troponin and myosin binding protein C. A focus of the troponin work has been to understand the isoform differences between the skeletal and cardiac isoforms of this major calcium receptor in muscle. In the case of myosin binding protein C, significant data are accumulating, indicating that this protein may act to modulate the primary calcium signals from troponin, and interest in its biological role has grown because of linkages between gene mutations in the cardiac isoform and serious heart disease. (C) 2011 Wiley Periodicals, Inc. Biopolymers 95: 505-516, 2011.
  • No Thumbnail Available
    Item
    K7del is a common TPM2 gene mutation associated with nemaline myopathy and raised myofibre calcium sensitivity
    (Oxford University Press, 2012-11-15) Mokbel, N; Ilkovski, B; Kreissl, M; Memo, M; Jeffries, CM; Marttila, M; Lehtokari, VL; Lemola, E; Grönholm, M; Yang, N; Menard, D; Marcorelles, P; Echaniz-Laguna, A; Reimann, J; Vainzof, M; Monnier, N; Ravenscroft, G; McNamara, E; Nowak, KJ; Laing, NG; Wallgren-Pettersson, C; Trewhella, J; Marston, S; Ottenheijm, C; North, KN; Clarke, NF
    Mutations in the TPM2 gene, which encodes β-tropomyosin, are an established cause of several congenital skeletal myopathies and distal arthrogryposis. We have identified a TPM2 mutation, p.K7del, in five unrelated families with nemaline myopathy and a consistent distinctive clinical phenotype. Patients develop large joint contractures during childhood, followed by slowly progressive skeletal muscle weakness during adulthood. The TPM2 p.K7del mutation results in the loss of a highly conserved lysine residue near the N-terminus of β-tropomyosin, which is predicted to disrupt head-to-tail polymerization of tropomyosin. Recombinant K7del-β-tropomyosin incorporates poorly into sarcomeres in C2C12 myotubes and has a reduced affinity for actin. Two-dimensional gel electrophoresis of patient muscle and primary patient cultured myotubes showed that mutant protein is expressed but incorporates poorly into sarcomeres and likely accumulates in nemaline rods. In vitro studies using recombinant K7del-β-tropomyosin and force measurements from single dissected patient myofibres showed increased myofilament calcium sensitivity. Together these data indicate that p.K7del is a common recurrent TPM2 mutation associated with mild nemaline myopathy. The p.K7del mutation likely disrupts head-to-tail polymerization of tropomyosin, which impairs incorporation into sarcomeres and also affects the equilibrium of the troponin/tropomyosin-dependent calcium switch of muscle. Joint contractures may stem from chronic muscle hypercontraction due to increased myofibrillar calcium sensitivity while declining strength in adulthood likely arises from other mechanisms, such as myofibre decompensation and fatty infiltration. These results suggest that patients may benefit from therapies that reduce skeletal muscle calcium sensitivity, and we highlight late muscle decompensation as an important cause of morbidity. © 2012, Oxford University Press
  • No Thumbnail Available
    Item
    Ligand-induced conformational changes via flexible linkers in the amino-terminal region of the inositol 1,4,5-trisphosphate receptor.
    (Elsevier, 2007-11-09) Chan, J; Whitten, AE; Jeffries, CM; Bosanac, I; Mal, TK; Ito, J; Porumb, H; Michikawa, T; Mikoshiba, K; Trewhella, J; Ikura, M
    Cytoplasmic Ca2+ signals are highly regulated by various ion transporters, including the inositol 1,4,5-trisphosphate (IP3) receptor (IP3), which functions as a Ca2+ release channel on the endoplasmic reticulum membrane. Crystal structures of the two N-terminal regulatory regions from type 1 IP3R have been reported; those of the IP3-binding core (IP3RCORE) with bound IP3, and the suppressor domain. This study examines the structural effects of ligand binding on an IP3R construct, designated IP3RN, that contains both the IP3-binding core and the suppressor domain. Our circular dichroism results reveal that the IP3- bound and IP3-free states have similar secondary structure content, consistent with preservation of the overall fold within the individual domains. Thermal denaturation data show that, while IP3 has a large effect on the stability of IP3RCORE, it has little effect on IP3RN, indicating that the suppressor domain is critical to the stability of IP3RN. The NMR data for IP3RN provide evidence for chemical exchange, which may be due to protein conformational dynamics in both apo and IP3-bound states: a conclusion supported by the small-angle X-ray scattering data. Further, the scattering data show that IP3RN undergoes a change m average conformation in response to IP3-binding and the presence of Ca2+, in the solution. Taken together, these data lead us to propose that there are two flexible linkers in the N-terminal region of lP(3)R that join stably folded domains and give rise to an equilibrium mixture of conformational sub-states containing compact and more extended structures. IP3 binding drives the conformational equilibrium toward more compact structures, while the presence of Ca2+ drives it to a more extended set. © 2007, Elsevier Ltd.
  • No Thumbnail Available
    Item
    LIM domain binding proteins 1 and 2 have different oligomeric states
    (Elsevier, 2010-05-28) Cross, AJ; Jeffries, CM; Trewhella, J; Matthews, JM
    LIM domain binding (Ldb) proteins are important regulators of LIM homeodomain and LIM-only proteins that specify cell fate in many different tissues. An essential feature of these proteins is the ability to self-associate, but there have been no studies that characterise the nature of this self-association. We have used deletion mutagenesis with yeast two-hybrid analysis to define the minimal self-association domains of Ldb1 and Ldb2 as residues 14–200 and 21–197, respectively. We then used a range of different biophysical methods, including sedimentation equilibrium and small-angle X-ray scattering to show that Ldb114–200 forms a trimer and Ldb221–197 undergoes a monomer–tetramer–octamer equilibrium, where the association in each case is of moderate affinity (105 M− 1). These modes of association represent a clear physical difference between these two proteins that otherwise appear to have very similar properties. The levels of association are more complex than previously assumed and emphasise roles of avidity and DNA looping in transcriptional regulation by Ldb1/LIM protein complexes. The abilities of Ldb1 and Ldb2 to form trimers and higher oligomers, respectively, should be considered in models of transcriptional regulation by Ldb1-containing complexes in a wide range of biological processes. © 2010, Elsevier Ltd.
  • No Thumbnail Available
    Item
    Macromolecular architecture of extracellular domain of αNRXN1: domain organization, flexibility, and insights into trans-synaptic disposition.
    (Elsevier (Cell Press), 2010-08-11) Comoletti, D; Miller, MT; Jeffries, CM; Wilson, J; Demeler, B; Taylor, P; Trewhella, J; Nakagawa, T
    Neurexins are multidomain synaptic cell-adhesion proteins that associate with multiple partnering proteins. Genetic evidence indicates that neurexins may contribute to autism, schizophrenia, and nicotine dependence. Using analytical ultracentrifugation, single-particle electron microscopy, and solution X-ray scattering, we obtained a three-dimensional structural model of the entire extracellular domain of neurexin-1α. This protein adopts a dimensionally asymmetric conformation that is monomeric in solution, with a maximum dimension of ~ 170 Å. The extracellular domain of α-neurexin maintains a characteristic “Y” shape, whereby LNS domains 1–4 form an extended base of the “Y” and LNS5-6 the shorter arms. Moreover, two major regions of flexibility are present: one between EGF1 and LNS2, corresponding to splice site 1, another between LNS5 and 6. We thus provide the first structural insights into the architecture of the extracellular region of neurexin-1α, show how the protein may fit in the synaptic cleft, and how partnering proteins could bind simultaneously. © 2010, Cell Press
  • Thumbnail Image
    Item
    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.
  • No Thumbnail Available
    Item
    Picornaviral loop-to-loop replication complex.
    (Elsevier, 2009-06) Claridge, JK; Headey, SJ; Chow, JYH; Schwalbe, M; Edwards, PJ; Jeffries, CM; Venugopal, H; Trewhella, J; Pascal, SM
    Picornaviruses replicate their RNA genomes through a highly conserved mechanism that involves an interaction between the principal viral protease (3C(pro)) and the 5'-UTR region of the viral genome. The 3C(pro) catalytic site is the target of numerous replication inhibitors. This paper describes the first structural model of a complex between a picornaviral 3C(pro) and a region of the 5'-UTR, stem-loop D (SLD). Using human rhinovirus as a model system, we have combined NMR contact information, small-angle X-ray scattering (SAXS) data, and previous mutagenesis results to determine the shape, position and relative orientation of the 3C(pro) and SLD components. The results clearly identify a 1:1 binding stoichiometry, with pronounced loops from each molecule providing the key binding determinants for the interaction. Binding between SLD and 3C(pro) induces structural changes in the proteolytic active site that is positioned on the opposite side of the protease relative to the RNA/protein interface, suggesting that subtle conformational changes affecting catalytic activity are relayed through the protein. © 2009, Elsevier Ltd.
  • Thumbnail Image
    Item
    Production and characterisation of modularly deuterated UBE2D1-Ub conjugate by small angle neutron and X-ray scattering
    (Springer, 2022-10-26) Pietras, Z; Duff, AP; Morad, V; Wood, K; Jeffries, CM; Sunnerhagen, M
    This structural study exploits the possibility to use modular protein deuteration to facilitate the study of ubiquitin signalling, transfer, and modification. A protein conjugation reaction is used to combine protonated E2 enzyme with deuterated ubiquitin for small angle X-ray and neutron scattering with neutron contrast variation. The combined biomolecules stay as a monodisperse system during data collection in both protonated and deuterated buffers indicating long stability of the E2–Ub conjugate. With multiphase ab initio shape restoration and rigid body modelling, we reconstructed the shape of a E2–Ub-conjugated complex of UBE2D1 linked to ubiquitin via an isopeptide bond. Solution X-ray and neutron scattering data for this E2–Ub conjugate in the absence of E3 jointly indicate an ensemble of open and backbent states, with a preference for the latter in solution. The approach of combining protonated and labelled proteins can be used for solution studies to assess localization and movement of ubiquitin and could be widely applied to modular Ub systems in general. © The Author(s) 2022. Open Access CC-BY
  • Thumbnail Image
    Item
    A round‐robin approach provides a detailed assessment of biomolecular small‐angle scattering data reproducibility and yields consensus curves for benchmarking
    (International Union of Crystallography (IUCr), 2022-11) Trewhella, J; Vachette, P; Bierma, J; Blanchet, Cl; Brookes, E; Chakravarthy, S; Chatzimagas, L; Cleveland, TE; Cowieson, NP; Crossett, B; Duff, AP; Franke, D; Gabel, F; Gillilan, RE; Graewert, MA; Grishaev, A; Guss, JM; Hammel, M; Hopkins, JB; Huang, Q; Hub, JS; Hura, GL; Irving, TC; Jeffries, CM; Jeong, C; Kirby, N; Krueger, S; Martel, A; Matsui, T; Li, N; Pérez, J; Porcar, L; Prangé, T; Rajkovic, I; Rocco, M; Rosenberg, DJ; Ryan, TM; Seifert, S; Sekiguchi, H; Svergun, D; Teixeira, S; Thureau, A; Weiss, TM; Whitten, AE; Wood, K; Zuo, X
    Through an expansive international effort that involved data collection on 12 small-angle X-ray scattering (SAXS) and four small-angle neutron scattering (SANS) instruments, 171 SAXS and 76 SANS measurements for five proteins (ribonuclease A, lysozyme, xylanase, urate oxidase and xylose isomerase) were acquired. From these data, the solvent-subtracted protein scattering profiles were shown to be reproducible, with the caveat that an additive constant adjustment was required to account for small errors in solvent subtraction. Further, the major features of the obtained consensus SAXS data over the q measurement range 0–1 Å−1 are consistent with theoretical prediction. The inherently lower statistical precision for SANS limited the reliably measured q-range to <0.5 Å−1, but within the limits of experimental uncertainties the major features of the consensus SANS data were also consistent with prediction for all five proteins measured in H2O and in D2O. Thus, a foundation set of consensus SAS profiles has been obtained for benchmarking scattering-profile prediction from atomic coordinates. Additionally, two sets of SAXS data measured at different facilities to q > 2.2 Å−1 showed good mutual agreement, affirming that this region has interpretable features for structural modelling. SAS measurements with inline size-exclusion chromatography (SEC) proved to be generally superior for eliminating sample heterogeneity, but with unavoidable sample dilution during column elution, while batch SAS data collected at higher concentrations and for longer times provided superior statistical precision. Careful merging of data measured using inline SEC and batch modes, or low- and high-concentration data from batch measurements, was successful in eliminating small amounts of aggregate or interparticle interference from the scattering while providing improved statistical precision overall for the benchmarking data set. © The Authors published by International Union of Crystallography. Open Access CC-By licence 4.0.
  • No Thumbnail Available
    Item
    Small-angle x-ray scattering reveals the N-terminal domain organization of cardiac myosin binding protein C
    (Elsevier, 2008-04-04) Jeffries, CM; Whitten, AE; Harris, SP; Trewhella, J
    Myosin binding protein C (MyBP-C) is a multidomain accessory protein of striated muscle sarcomeres. Three domains at the N-terminus of MyBP-C (Cl-m-C2) play a crucial role in maintaining and modulating actomyosin interactions. The cardiac isoform has an additional N-terminal domain (CO) that is postulated to provide a greater level of regulatory control in cardiac muscle. We have used small-angle X-ray scattering, ab initio shape restoration, and rigid-body modeling to determine the average shape and spatial arrangement of the four N-terminal domains of cardiac MyBP-C (C0C2) and a three-domain variant that is analogous to the N-terminus of the skeletal isoform (C1C2). We found that the domains of both proteins are tandemly arranged in a highly extended configuration that is sufficiently long to span the interfilament cross-bridge distances in vivo and, hence, be poised to modulate these interactions. The average spatial organization of the C1, m, and C2 domains is not significantly perturbed by the removal of the cardiac-specific CO domain, suggesting that the interdomain interfaces, while relatively small in area, have a degree of rigidity. Modeling the C0C2 and C1C2 scattering data reveals that the structures of the C0 and m domains (also referred to as the 'MyBP motif') are compact and have dimensions that are consistent with the immunoglobulin fold superfamily of proteins. Sequence analysis, homology modeling, and circular dichroism experiments support the conclusion that the previously undetermined structures of these domains can be characterized as having an immunoglobulin-like fold. Atomic models using the known NMR structures for C1 and C2 as well as homology models for the C0 and m domains provide insights into the placement of conserved serine residues of the m domain that are phosphorylated in vivo and cause a change in muscle fiber contraction by abolishing interactions with myosin. © 2008, Elsevier Ltd.
  • Thumbnail Image
    Item
    Solution 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, JM
    Lhx3 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.
  • No Thumbnail Available
    Item
    Solution structure studies of monomeric human TIP47/perilipin-3 reveal a highly extended conformation
    (Wiley, 2012-04-17) Hynson, RMG; Jeffries, CM; Trewhella, J; Cocklin, S
    Tail-interacting protein of 47 kDa (TIP47) has two putative functions: lipid biogenesis and mannose 6-phosphate receptor recycling. Progress in understanding the molecular details of these two functions has been hampered by the lack of structural data on TIP47, with a crystal structure of the C-terminal domain of the mouse homolog constituting the only structural data in the literature so far. Our studies have first provided a strategy to obtain pure monodisperse preparations of the full-length TIP47/perilipin-3 protein, as well as a series of N-terminal truncation mutants with no exogenous sequences. These constructs have then enabled us to obtain the first structural characterization of the full-length protein in solution. Our work demonstrates that the N-terminal region of TIP47/perilipin-3, in contrast to the largely helical C-terminal region, is predominantly beta-structure with turns and bends. Moreover, we show that full-length TIP47/perilipin-3 adopts an extended conformation in solution, with considerable spatial separation of the N- and C-termini that would likely translate into a separation of functional domains. Proteins 2012;. © 2012, Wiley-Blackwell.