ANSTO :: Browsing by Author "Whitten, AE"

Browsing by Author "Whitten, AE"

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The ANSTO contribution to a project to provide experimental standards for SAS profile prediction
(Australian Institure of Nuclear Sicence and Engineering (AINSE), 2020-11-11) Duff, AP; Kirby, N; Ryan, T; Trewhella, J; Whitten, AE; Wood, K
The program CRYSOL1, was the first method developed to rapidly calculate small-angle scattering (SAS) profiles from atomic coordinates of biomolecules. It was a major breakthrough, providing the missing link between high-resolution structures and solution SAS data. The importance of this breakthrough is evident in the fact that decades later, alternate methods continue to be published with various claims of improvement. To date, each of the alternate methods published have been validated using different data sets and models. A consensus set of high quality data would be of considerable value in benchmarking the different approaches. In order to evaluate different approaches to including the hydration layer contribution to the SAS profile, it is desirable to have data obtained using X-rays (SAXS) and neutrons (SANS), the latter in H2O and D2O, as the hydration layer contribution differs significantly for each of these measurements. To develop a consensus set of high quality data, an international project involving the efforts of 37 researchers from 11 different X-ray and 3 different neutron scattering facilities across Asia, Europe and North Ⓒ The Authors.
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
Crystallographic characterization of fluorapatite glass-ceramics synthesized from industrial waste
(Cambridge University Press, 2017-09-15) Loy, CW; Matori, KA; Zainuddin, N; Whitten, AE; Rehm, C; de Campo, L; Sokolova, AV; Schmid, S
A series of phase transformations of a novel fluoroaluminosilicate glass forming a range of fluorapatite glass-ceramics on sintering are reported. The sintering process induces formation of fluorapatite, mullite, and anorthite phases within the amorphous silicate matrices of the glass-ceramics. The fluoroaluminosilicate glass, SiO2–Al2O3–P2O5–CaO–CaF2, is prepared from waste materials, such as rice husk ash, pacific oyster shells, and disposable aluminium cans. The thermally induced crystallographic and microstructure evolution of the fluoroaluminosilicate glass towards the fluorapatite glass-ceramics, with applications in dental and bone restoration, are investigated by powder X-ray diffraction and small-angle neutron-scattering techniques. © Cambridge University Press.
Evolution of quaternary structure in a homotetrameric enzyme
(Elsevier, 2008-07-18) Griffin, MDW; Dobson, RCJ; Pearce, FG; Antonio, L; Whitten, AE; Liew, K; Mackay, JP; Trewhella, J; Jameson, GB; Perugini, MA; Gerrard, JA
Dihydrodipicolinate synthase (DHDPS) is an essential enzyme in (S)-lysine biosynthesis and an important antibiotic target. All X-ray crystal structures solved to date reveal a homotetrameric enzyme. In order to explore the role of this quaternary structure, dimeric variants of Escherichia coli DHDPS were engineered and their properties were compared to those of the wild-type tetrameric form. X-ray crystallography reveals that the active site is not disturbed when the quaternary structure is disrupted. However, the activity of the dimeric enzymes in solution is substantially reduced, and a tetrahedral adduct of a substrate analogue is observed to be trapped at the active site in the crystal form. Remarkably, heating the dimeric enzymes increases activity. We propose that the homotetrameric structure of DHDPS reduces dynamic fluctuations present in the dimeric forms and increases specificity for the first substrate, pyruvate. By restricting motion in a key catalytic motif, a competing, non-productive reaction with a substrate analogue is avoided. Small-angle X-ray scattering and mutagenesis data, together with a B-factor analysis of the crystal structures, support this hypothesis and lead to the suggestion that in at least some cases, the evolution of quaternary enzyme structures might serve to optimise the dynamic properties of the protein subunits. © 2008, Elsevier Ltd.
Hydration processes in tricalcium silicate: application of the boundary nucleation model to quasielastic neutron scattering data.
(American Chemical Society, 2009-02-12) Peterson, VK; Whitten, AE
Tricalcium silicate hydration is followed using quasielastic neutron scattering (QENS) and the kinetics of product nucleation and growth modeled using a boundary nucleation (BN) model. The BN model, previously applied once to calorimetry data of hydrating tricalciurn silicate, is applied to QENS data that includes components that do not follow the evolution of heat (measured using calorimetry). Variations in modeling conditions are explored, including combination with a second model describing later, diffusion-limited, behavior. The BN model produces comparable quality fits to those from the commonly used Avrami-derived model and is found to support the theory that nucleation and growth begins at the time of mixing and proceeds with a single kinetic process. © 2009, American Chemical Society
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.
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.
Low resolution structural studies of Munc18c complexed with a Syntaxin-4/T4- Lysozyme Fusion
(Australian Institute of Nuclear Science and Engineering, 2016-11-29) Whitten, AE; Rehman, AU; Hu, SH; Tnimov, Z; Christie, MP; King, GJ; Jarrott, RJ; Norwood, S; Alexandrov, K; Collins, BM; Martin, JL
Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNARE) act at every intracellular trafficking pathway. Cognate v-SNAREs (e.g. VAMP) and t-SNAREs (Syntaxin (Sx) and SNAP) form a high affinity SNARE ternary complex (Sx-SNAP-VAMP) that brings the membranes together, triggering fusion. Syntaxins consist of a SNARE motif, and a three-helix bundle. In an open confirmation, the SNARE motif is free to form the SNARE ternary complex (stimulating fusion), but in the closed confirmation fusion is inhibited. Sec1p/Munc18 (SM) proteins bind to Sx, regulating SNARE mediated fusion [1], but their exact role is not well understood [2-4]. In the cell, Sx is bound to the membrane, and it is possible that this tethering may influence the manner in which it interacts with other proteins. As a means of investigating structural changes arising due to tethering, here, we investigate how the addition of a C-terminal T4-Lysozyme (soluble) fusion to Sx4 modulates its interaction with Munc18c. Preliminary low-resolution models of the Munc18c-Sx4T4 complex optimized against small-angle scattering data will be presented.
Low-resolution solution structures of Munc18:Syntaxin protein complexes indicate an open binding mode driven by the Syntaxin N-peptide
(National Academy of Sciences, 2012-06-19) Christie, MP; Whitten, AE; King, GJ; Hu, SH; Jarrott, RJ; Chen, KE; Duff, AP; Callow, P; Collins, BM; James, DE; Martin, JL
When nerve cells communicate, vesicles from one neuron fuse with the presynaptic membrane releasing chemicals that signal to the next. Similarly, when insulin binds its receptor on adipocytes or muscle, glucose transporter-4 vesicles fuse with the cell membrane, allowing glucose to be imported. These essential processes require the interaction of SNARE proteins on vesicle and cell membranes, as well as the enigmatic protein Munc18 that binds the SNARE protein Syntaxin. Here, we show that in solution the neuronal protein Syntaxin1a interacts with Munc18-1 whether or not the Syntaxin1a N-peptide is present. Conversely, the adipocyte protein Syntaxin4 does not bind its partner Munc18c unless the N-peptide is present. Solution-scattering data for the Munc18-1:Syntaxin1a complex in the absence of the N-peptide indicates that this complex adopts the inhibitory closed binding mode, exemplified by a crystal structure of the complex. However, when the N-peptide is present, the solution-scattering data indicate both Syntaxin1a and Syntaxin4 adopt extended conformations in complexes with their respective Munc18 partners. The low-resolution solution structure of the open Munc18:Syntaxin binding mode was modeled using data from cross-linking/mass spectrometry, small-angle X-ray scattering, and small-angle neutron scattering with contrast variation, indicating significant differences in Munc18:Syntaxin interactions compared with the closed binding mode. Overall, our results indicate that the neuronal Munc18-1:Syntaxin1a proteins can adopt two alternate and functionally distinct binding modes, closed and open, depending on the presence of the N-peptide, whereas Munc18c:Syntaxin4 adopts only the open binding mode. © 2012, National Academy of Sciences.
Microstructural evolution of dental glass-ionomer cements during setting reaction followed using SANS and USANS
(International Conference on Neutron Scattering, 2017-07-12) Loy, CW; Matori, KA; Zainuddin, N; Whitten, AE; Rehm, C; de Campo, L; Schmid, S
Glass-ionomer cement (GIC) is a biocompatible material which is clinically used for dental filling. The main challenges for further developing GIC in dental applications are improving the mechanical strength and controlling the setting reaction. During the setting reaction, poly (acrylic acid) attacks the fluoroaluminosilicate glass particles to form a siliceous hydrogel layer, glass core and polyalkenoate matrix in paste form. The siliceous hydrogel layer undergoes dehydration to yield a strong cross-linkage to bind both polymer and glass particles into a cement structure. This study presents the application of small angle neutron scattering (SANS) and ultra small angle neutron scattering (USANS) with contrast variation techniques to study the microstructure evolution of a complex GIC paste during 48 hours of the setting reaction. A few GIC pastes are prepared from medical grade poly (acrylic acid), SiO2–Al2O3–P2O5–Na2O–CaO–CaF2-based fluoroaluminosilicate glasses and a mixture of H2O:D2O solvent following the ISO9917-1:2007 cement preparation method. The combination of SANS (Bilby@ACNS) and USANS (Kookaburra@ACNS) provides microstructure information of GIC paste over the length scale of 1 nm to 10 µm. The microstructure change of each phase in GIC pastes is investigated at different contrast conditions by varying the H2O:D2O ratio for both neutron scattering experiments. The macro- and nano-scale features of the polymer-glass-hydrogel phases in GIC paste during the setting reaction as well as their impact on mechanical strengths are presented in this study.
MULCh: modules for the analysis of small-angle neutron contrast variation data from biomolecular assemblies.
(Wiley-Blackwell, 2008-02) Whitten, AE; Ca, SZ; Trewhella, J
Small-angle neutron scattering with contrast variation can fill important gaps in our understanding of biomolecular assemblies, providing constraints that can aid in the construction of molecular models and in subsequent model refinements. This paper describes the implementation of simple tools for analysing neutron contrast variation data, accessible via a user-friendly web-based interface (http://www.mmb.usyd.edu.au/NCVWeb/). There are three modules accessible from the website to analyse neutron contrast variation data from bimolecular complexes. The first module, Contrast, computes neutron contrasts of each component of the complex required by the other two modules; the second module, R, analyses the contrast dependence of the radii of gyration to yield information relating to the size and disposition of each component in the complex; and the third, Compost, decomposes the contrast variation series into composite scattering functions, which contain information regarding the shape of each component of the complex, and their orientation with respect to each other. © 2008, Wiley-Blackwell.
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.
Oil-in-water emulsion system stabilized by emulsion droplets coated with whey protein microgels
(Australian Institute of Nuclear Science and Engineering (AINSE), 2020-11-11) Cheng, LR; Ye, AJ; Hemar, Y; Single, H; Gilbert, EP; de Campo, L; Whitten, AE
Structurally designed emulsions are a developing group that is likely to find increasing utilization within the food industry because of their potential advantages over conventional emulsions. A novel droplet-stabilized (DS) emulsion system emulsified with casein micelles has been previously reported [1]. However, the mechanism of DS emulsion formation, physicochemical properties, and stability are not yet been fully explored. In the present study, heat-induced whey protein microgel (WPM) particles were used as an alternative emulsifying agent. The structure of WPM particles on the formation and physicochemical properties of the primary (PE) and the DS emulsions was investigated [2]. WPM particles were prepared by heating 4 wt% whey protein isolate solution in the presence (PB) or absence (NPB) of 10 mM phosphate buffer at pH 5.9, 85°C for 45 min, followed by washing, centrifugation, and micro-fluidization. The PE coated with WPM was homogenized using 3 passes at the pressure of 250/50 bar. DS emulsions were prepared by mixing (at 30000 rev/min for 2 min) 10 wt% oil with 10, 30, or 60 wt% PE. The structure of WPM particles and emulsions were analyzed by dynamic light scattering, confocal light scattering microscopy (CLSM), transmission electronic microscopy (TEM), and the combination of small and ultra-small angle neutron scattering (SANS and USANS). The results showed that the WPM particles produced in the absence of phosphate buffer (WPM-NPB) were smooth spherical particles, giving a surface fractal dimension of 2.0 and a hydrodynamic diameter of 270 nm. However, WPM particles made in the presence of phosphate buffer (WPM-PB) were rough spherical particles with a surface fractal dimension of 2.3 and a hydrodynamic diameter of 290 nm. Particle fragments present in the WPM-PB dispersion, resulted in their competitive adsorption onto the surface of the DS emulsions; reducing the adsorption of PE droplets. For the PE coated with WPM-NPB particles (PE-NPB), flocculation due to protein bridging and protein intramolecular interaction, lead to a network with a fractal dimension of 2.7. For the DS emulsions stabilized by PE-NPB, the interfacial layer thickness of DS emulsion droplets increased with the increase in the concentration of PE as observed by CLSM, whereas the size of DS emulsion droplets decreased. A fractal network consisting of adsorbed PE droplets on the interfacial layer of DS emulsion was observed by TEM and measured by USANS with a fractal dimension of 3.0, suggesting a very rough interface. These results suggest that both the structure of the interfacial layer and the size of the DS emulsion is dependent on the concentration of the PE used.
SANS time-of-flight instrument Bilby at ACNS, ANSTO
(Australian Institute of Nuclear Science and Engineering (AINSE), 2020-11-11) Sokolova, AV; Whitten, AE; de Campo, L
ANSTO for more than ten years successfully operates Small Angle Neutron Scattering (SANS) instrument Quokka [1] and in 2016 commenced user operation of the second SANS instrument, Bilby [2]. Ultra-small angle scattering machine Kookaburra [3] is completing the set of the SANS instruments at ANSTO. Bilby exploits neutron Time-of-Flight (ToF) to extend the measurable Q-range, over and above what is possible on a conventional reactor-based monochromatic SANS instrument. In ToF mode, the choppers are used to create neutron pulses of variable (~3% ‒ 30%) wavelength resolution. Two arrays of position-sensitive detectors in combination with utilizing of wide wavelength range provide the capability to collect scattering data of wide angular diapason without changing the experimental set-up (maximum accessible Q on the instrument is 0.001-1.8Å-1). Additionally to the ToF, Bilby can operate in monochromatic mode. The question is how the advanced design features can be applied to the real scientific questions. In short, having a large dynamic range available in one go opens up a possibility to study complex systems like micelles and hierarchical materials. Additionally, there is a range of sample environments available allowing to change conditions in situ, which is priceless for study a range of samples stretching from colloids to metals. In my presentation, I will be giving several examples demonstrating how ToF SANS can bring light to structural changes of the surfactant wormlike micelles structure under various conditions. Some cases will be presented to show that the monochromatic mode is also the one producing valuable results. The main accent will be made on a recently published work, done solely on Bilby along with examples of combining SANS and USANS techniques. © The authors.
Small angle neutron scattering study of a gehlenite-based ceramic fabricated from industrial waste
(Trans Tech Publications, 2017-11-13) Loy, CW; Matori, K.A; Zainuddin, N; Whitten, AE; de Campo, L; Nasir, NIM; Pallan, NFB; Zaid, MHM; Alassan, ZN; Schmid, S
This paper presents a small angle neutron scattering (SANS)study of a novel porous gehlenite-based ceramic, synthesised from a homogeneous powder mixture of soda-lime-silicate (SLS)glass,α-alumina, calcite and calcium fluoride via solid-state sintering at 1200 °C. The products of sintering at single temperatures from 600 to 1200 °C are examined by X-ray diffraction (XRD). Sintering of the mixture below1200 °C forms two intermediate phases (Na2CaSi3O8and Ca4Si2O7F2). Nepheline and α-alumina are minor phases in the gehlenite-based ceramic fabricated through sintering at 1200 °C. The microstructure of the gehlenite-based ceramic is investigated using field-emission scanning electron microscopy (FESEM) and SANS at the Australian Centre for Neutron Scattering. This study also evaluated the specific surface area of the gehlenite-based ceramic (~3.0 m2cm–3) from quantitative analysis of SANS data. ©2019 Trans Tech Publications, Switzerland
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.
SNARE-ing the structures of Sec1/Munc18 proteins
(Elsevier BV, 2014-12-01) Archbold, JK; Whitten, AE; Hu, SH; Collins, BM; Martin, JL
Membrane fusion is essential for cellular transport in eukaryotes. Abnormalities contribute to a wide range of diseases including diabetes and neurological disorders. A key regulator of SNARE-mediated membrane fusion is the Sec1/Munc18 (SM) protein family. Universal structural features of SM proteins have been identified that affect the way these interact with their partner Syntaxin SNARE proteins. Whilst the molecular basis for SM-regulated SNARE complex formation has been extensively studied, it remains poorly understood. Recent crystal structures of SM proteins alone or in complex have provided new insight. Here we examine the available structural information on SM proteins for clues to how these enigmatic proteins might regulate SNARE complex assembly and membrane fusion. © 2014, Elsevier Ltd.
Solution structure of ectodomains of the insulin receptor family: the ectodomain of the type 1 insulin-like growth factor receptor displays asymmetry of ligand binding accompanied by limited conformational change
(Elsevier, 2009-12-18) Whitten, AE; Smith, BJ; Menting, JG; Margetts, MB; McKern, NM; Lovrecz, GO; Adams, TE; Richards, K; Bentley, JD; Trewhella, J; Ward, CW; Lawrence, MC
The insulin receptor (IR) and the homologous Type 1 insulin-like growth factor receptor (IGF-1R) are cell-surface tyrosine kinase receptors that effect signaling within the respective pathways of glucose metabolism and normal human growth. While ligand binding to these receptors is assumed to result in a structural transition within the receptor ectodomain that then effects signal transduction across the cell membrane, little is known about the molecular detail of these events. Presented here are small-angle X-ray scattering data obtained from the IR and IGF-1R ectodomains in solution. We show that, in solution, the ectodomains of IR and IGF-1R have a domain disposition that is very similar to that seen in the crystal structure of the ectodomain of IR, despite the constituent domains being in relatively sparse contact and potentially mobile. We also show that the IGF-1R ectodomain is capable of binding up to three molecules of IGF-1 in solution, with surprisingly little apparent change in relative domain disposition compared to the apo, form. While the observed 3:1 ligand-binding stoichiometry appears to contradict earlier explanations of the absence of a bell-shaped dose-response curve for IGF-1R in ligand displacement assays, it is readily understood in the context of the harmonic oscillator model of the negative cooperativity of ligand binding to IGF-1R. Taken together, our findings suggest that the structural movements within these receptors upon ligand binding are small and are possibly limited to local rotation of domains. © 2009, Elsevier Ltd.
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.
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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