Publications by authors named "Jill Trewhella"

87 Publications

Contrasting DNA-binding behaviour by ISL1 and LHX3 underpins differential gene targeting in neuronal cell specification.

J Struct Biol X 2021 15;5:100043. Epub 2020 Dec 15.

School of Life and Environmental Sciences, University of Sydney, NSW 2006, Australia.

The roles of ISL1 and LHX3 in the development of spinal motor neurons have been well established. Whereas LHX3 triggers differentiation into interneurons, the additional expression of ISL1 in developing neuronal cells is sufficient to redirect their developmental trajectory towards spinal motor neurons. However, the underlying mechanism of this action by these transcription factors is less well understood. Here, we used electrophoretic mobility shift assays (EMSAs) and surface plasmon resonance (SPR) to probe the different DNA-binding behaviours of these two proteins, both alone and in complexes mimicking those found in developing neurons, and found that ISL1 shows markedly different binding properties to LHX3. We used small angle X-ray scattering (SAXS) to structurally characterise DNA-bound species containing ISL1 and LHX3. Taken together, these results have allowed us to develop a model of how these two DNA-binding modules coordinate to regulate gene expression and direct development of spinal motor neurons.
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http://dx.doi.org/10.1016/j.yjsbx.2020.100043DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7797366PMC
December 2020

Federating Structural Models and Data: Outcomes from A Workshop on Archiving Integrative Structures.

Structure 2019 12 25;27(12):1745-1759. Epub 2019 Nov 25.

Department of Pharmaceutical Chemistry, University of California, San Francisco, CA 94158, USA; Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, San Francisco, CA 94158, USA; California Institute for Quantitative Biosciences, University of California, San Francisco, San Francisco, CA 94158, USA. Electronic address:

Structures of biomolecular systems are increasingly computed by integrative modeling. In this approach, a structural model is constructed by combining information from multiple sources, including varied experimental methods and prior models. In 2019, a Workshop was held as a Biophysical Society Satellite Meeting to assess progress and discuss further requirements for archiving integrative structures. The primary goal of the Workshop was to build consensus for addressing the challenges involved in creating common data standards, building methods for federated data exchange, and developing mechanisms for validating integrative structures. The summary of the Workshop and the recommendations that emerged are presented here.
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http://dx.doi.org/10.1016/j.str.2019.11.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7108332PMC
December 2019

E3 ubiquitin-protein ligase TRIM21-mediated lysine capture by UBE2E1 reveals substrate-targeting mode of a ubiquitin-conjugating E2.

J Biol Chem 2019 07 3;294(30):11404-11419. Epub 2019 Jun 3.

Department of Physics, Chemistry and Biology, Division of Chemistry, Linköping University, SE-58183 Linköping, Sweden

The E3 ubiquitin-protein ligase TRIM21, of the RING-containing tripartite motif (TRIM) protein family, is a major autoantigen in autoimmune diseases and a modulator of innate immune signaling. Together with ubiquitin-conjugating enzyme E2 E1 (UBE2E1), TRIM21 acts both as an E3 ligase and as a substrate in autoubiquitination. We here report a 2.82-Å crystal structure of the human TRIM21 RING domain in complex with the human E2-conjugating UBE2E1 enzyme, in which a ubiquitin-targeted TRIM21 substrate lysine was captured in the UBE2E1 active site. The structure revealed that the direction of lysine entry is similar to that described for human proliferating cell nuclear antigen (PCNA), a small ubiquitin-like modifier (SUMO)-targeted substrate, and thus differs from the canonical SUMO-targeted substrate entry. In agreement, we found that critical UBE2E1 residues involved in the capture of the TRIM21 substrate lysine are conserved in ubiquitin-conjugating E2s, whereas residues critical for SUMOylation are not conserved. We noted that coordination of the acceptor lysine leads to remodeling of amino acid side-chain interactions between the UBE2E1 active site and the E2-E3 direct interface, including the so-called "linchpin" residue conserved in RING E3s and required for ubiquitination. The findings of our work support the notion that substrate lysine activation of an E2-E3-connecting allosteric path may trigger catalytic activity and contribute to the understanding of specific lysine targeting by ubiquitin-conjugating E2s.
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http://dx.doi.org/10.1074/jbc.RA119.008485DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6663867PMC
July 2019

Mutation in a flexible linker modulates binding affinity for modular complexes.

Proteins 2019 05 9;87(5):425-429. Epub 2019 Mar 9.

School of Life and Environmental Sciences, University of Sydney, Sydney, New South Wales, Australia.

Tandem beta zippers are modular complexes formed between repeated linear motifs and tandemly arrayed domains of partner proteins in which β-strands form upon binding. Studies of such complexes, formed by LIM domain proteins and linear motifs in their intrinsically disordered partners, revealed spacer regions between the linear motifs that are relatively flexible but may affect the overall orientation of the binding modules. We demonstrate that mutation of a solvent exposed side chain in the spacer region of an LHX4-ISL2 complex has no significant effect on the structure of the complex, but decreases binding affinity, apparently by increasing flexibility of the linker.
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http://dx.doi.org/10.1002/prot.25675DOI Listing
May 2019

Archiving of Integrative Structural Models.

Adv Exp Med Biol 2018;1105:261-272

RCSB Protein Data Bank, Institute for Quantitative Biomedicine, Rutgers, The State University of New Jersey, Piscataway, NJ, USA.

Integrative or hybrid structural biology involves the determination of three-dimensional structures of macromolecular assemblies by combining information from a variety of experimental and computational methods. Archiving the results of integrative/hybrid modeling methods have complex requirements and existing archiving mechanisms are insufficient to handle these pre-requisites. Three concepts important for archiving integrative/hybrid models are presented in this chapter: (1) building a federated network of structural model and experimental data archives, (2) development of a common set of data standards, and (3) creation of mechanisms for interoperation and data exchange among the repositories in a federation. Methods proposed for achieving these objectives are also discussed.
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http://dx.doi.org/10.1007/978-981-13-2200-6_16DOI Listing
July 2019

Small Angle Scattering and Structural Biology: Data Quality and Model Validation.

Authors:
Jill Trewhella

Adv Exp Med Biol 2018;1105:77-100

School of Life and Environmental Sciences, The University of Sydney, NSW, Australia.

This chapter provides a brief review of the current state-of-the-art in small-angle scattering (SAS) from biomolecules in solution in regard to: (1) sample preparation and instrumentation, (2) data reduction and analysis, and (3) three-dimensional structural modelling and validation. In this context, areas of ongoing research in regard to the interpretation of SAS data will be discussed with a particular focus on structural modelling using computational methods and data from different experimental techniques, including SAS (hybrid methods). Finally, progress made in establishing community accepted publication guidelines and a standard reporting framework that includes SAS data deposition in a public data bank will be described. Importantly, SAS data with associated meta-data can now be held in a format that supports exchange between data archives and seamless interoperability with the world-wide Protein Data Bank (wwPDB). Biomolecular SAS is thus well positioned to contribute to an envisioned federation of data archives in support of hybrid structural biology.
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http://dx.doi.org/10.1007/978-981-13-2200-6_7DOI Listing
July 2019

Bayesian inference of protein conformational ensembles from limited structural data.

PLoS Comput Biol 2018 12 17;14(12):e1006641. Epub 2018 Dec 17.

Biochemistry and Structural Biology, University of Lund, Lund, Sweden.

Many proteins consist of folded domains connected by regions with higher flexibility. The details of the resulting conformational ensemble play a central role in controlling interactions between domains and with binding partners. Small-Angle Scattering (SAS) is well-suited to study the conformational states adopted by proteins in solution. However, analysis is complicated by the limited information content in SAS data and care must be taken to avoid constructing overly complex ensemble models and fitting to noise in the experimental data. To address these challenges, we developed a method based on Bayesian statistics that infers conformational ensembles from a structural library generated by all-atom Monte Carlo simulations. The first stage of the method involves a fast model selection based on variational Bayesian inference that maximizes the model evidence of the selected ensemble. This is followed by a complete Bayesian inference of population weights in the selected ensemble. Experiments with simulated ensembles demonstrate that model evidence is capable of identifying the correct ensemble and that correct number of ensemble members can be recovered up to high level of noise. Using experimental data, we demonstrate how the method can be extended to include data from Nuclear Magnetic Resonance (NMR) and structural energies of conformers extracted from the all-atom energy functions. We show that the data from SAXS, NMR chemical shifts and energies calculated from conformers can work synergistically to improve the definition of the conformational ensemble.
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http://dx.doi.org/10.1371/journal.pcbi.1006641DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6312354PMC
December 2018

Structural properties of a haemophore facilitate targeted elimination of the pathogen Porphyromonas gingivalis.

Nat Commun 2018 10 5;9(1):4097. Epub 2018 Oct 5.

School of Dentistry, Faculty of Medicine and Health, The University of Sydney, Camperdown, NSW, 2145, Australia.

Porphyromonas gingivalis is a keystone bacterial pathogen of chronic periodontitis. P. gingivalis is unable to synthesise the porphyrin macrocycle and relies on exogenous porphyrin, including haem or haem biosynthesis intermediates from host sources. We show that under the iron-limited conditions prevailing in tissue environments, P. gingivalis expresses a haemophore-like protein, HusA, to mediate the uptake of essential porphyrin and support pathogen survival within epithelial cells. The structure of HusA, together with titration studies, mutagenesis and in silico docking, show that haem binds in a hydrophobic groove on the α-helical structure without the typical iron coordination seen in other haemophores. This mode of interaction allows HusA to bind to a variety of abiotic and metal-free porphyrins with higher affinities than to haem. We exploit this unusual porphyrin-binding activity of HusA to target a prototypic deuteroporphyrin-metronidazole conjugate with restricted antimicrobial specificity in a Trojan horse strategy that effectively kills intracellular P. gingivalis.
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http://dx.doi.org/10.1038/s41467-018-06470-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6173696PMC
October 2018

The C-Terminal Zinc Fingers of ZBTB38 are Novel Selective Readers of DNA Methylation.

J Mol Biol 2018 02 27;430(3):258-271. Epub 2017 Dec 27.

Department of Chemistry, University of Utah, 315 South 1400 East, Salt Lake City, UT 84112, United States. Electronic address:

Methyl-CpG binding proteins play an essential role in translating DNA methylation marks into a downstream transcriptional response, which has implications for both normal cell function as well as disease. Although for many of these proteins, a detailed mechanistic understanding for how this cellular process is mediated remains to be determined. ZBTB38 is an under-characterized member of the zinc finger (ZF) family of methyl-CpG binding proteins. Functional knowledge has been gained for its conserved methylated DNA binding N-terminal ZF region; however, a specific role for the C-terminal set of five ZFs remains to be elucidated. Here we demonstrate for the first time that a subset of the C-terminal ZBTB38 ZFs exhibit high-affinity DNA interactions and that preferential targeting of the consensus DNA site is methyl specific. Utilizing a hybrid approach, a model for the C-terminal ZBTB38 ZFs in complex with its cognate DNA target is proposed, providing insight into a possible novel mode of methylated DNA recognition. Furthermore, it is shown that the C-terminal ZFs of ZBTB38 can directly occupy promoters harboring the newly identified sequence motif in cell in a methyl-dependent manner and, depending on the gene context, contribute to modulating transcriptional response. Combined, these findings provide evidence for a key and novel physiological function for the C-terminal ZF domain of ZBTB38.
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http://dx.doi.org/10.1016/j.jmb.2017.12.014DOI Listing
February 2018

PDB-Dev: a Prototype System for Depositing Integrative/Hybrid Structural Models.

Structure 2017 09;25(9):1317-1318

School of Life and Environmental Sciences, The University of Sydney, Sydney, NSW 2006, Australia; Department of Chemistry, University of Utah, Salt Lake City, UT 84112, USA.

Burley et al. (leadership of the Worldwide PDB [wwPDB] Partnership [wwpdb.org] and the wwPDB Integrative/Hybrid Methods Task Force) announce public release of a prototype system for depositing integrative/hybrid structural models, PDB-Development (PDB-Dev; https://pdb-dev.wwpdb.org).
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http://dx.doi.org/10.1016/j.str.2017.08.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5821105PMC
September 2017

2017 publication guidelines for structural modelling of small-angle scattering data from biomolecules in solution: an update.

Acta Crystallogr D Struct Biol 2017 Sep 18;73(Pt 9):710-728. Epub 2017 Aug 18.

ANSTO, New Illawarra Road, Lucas Heights, NSW 2234, Australia.

In 2012, preliminary guidelines were published addressing sample quality, data acquisition and reduction, presentation of scattering data and validation, and modelling for biomolecular small-angle scattering (SAS) experiments. Biomolecular SAS has since continued to grow and authors have increasingly adopted the preliminary guidelines. In parallel, integrative/hybrid determination of biomolecular structures is a rapidly growing field that is expanding the scope of structural biology. For SAS to contribute maximally to this field, it is essential to ensure open access to the information required for evaluation of the quality of SAS samples and data, as well as the validity of SAS-based structural models. To this end, the preliminary guidelines for data presentation in a publication are reviewed and updated, and the deposition of data and associated models in a public archive is recommended. These guidelines and recommendations have been prepared in consultation with the members of the International Union of Crystallography (IUCr) Small-Angle Scattering and Journals Commissions, the Worldwide Protein Data Bank (wwPDB) Small-Angle Scattering Validation Task Force and additional experts in the field.
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http://dx.doi.org/10.1107/S2059798317011597DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5586245PMC
September 2017

A Highly Conserved Yet Flexible Linker Is Part of a Polymorphic Protein-Binding Domain in Myosin-Binding Protein C.

Structure 2016 11 6;24(11):2000-2007. Epub 2016 Oct 6.

School of Life and Environmental Sciences, The University of Sydney, NSW 2006, Australia. Electronic address:

The nuclear magnetic resonance (NMR) structure of the tri-helix bundle (THB) of the m-domain plus C2 (ΔmC2) of myosin-binding protein C (MyBP-C) has revealed a highly flexible seven-residue linker between the structured THB and C2. Bioinformatics shows significant patterns of conservation across the THB-linker sequence, with the linker containing a strictly conserved serine in all MyBP-C isoforms. Clinically linked mutations further support the functional significance of the THB-linker region. NMR, small-angle X-ray scattering, and binding studies show the THB-linker plus the first ten residues of C2 undergo dramatic changes when ΔmC2 binds Ca-calmodulin, with the linker and C2 N-terminal residues contributing significantly to the affinity. Modeling of all available experimental data indicates that the THB tertiary structure must be disrupted to form the complex. These results are discussed in the context of the THB-linker and the N-terminal residues of C2 forming a polymorphic binding domain that could accommodate multiple binding partners in the dynamic sarcomere.
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http://dx.doi.org/10.1016/j.str.2016.08.018DOI Listing
November 2016

Mutation-Induced Population Shift in the MexR Conformational Ensemble Disengages DNA Binding: A Novel Mechanism for MarR Family Derepression.

Structure 2016 08 14;24(8):1311-1321. Epub 2016 Jul 14.

Division of Chemistry, Department of Physics, Chemistry and Biology, Linköping University, 581 83 Linköping, Sweden. Electronic address:

MexR is a repressor of the MexAB-OprM multidrug efflux pump operon of Pseudomonas aeruginosa, where DNA-binding impairing mutations lead to multidrug resistance (MDR). Surprisingly, the crystal structure of an MDR-conferring MexR mutant R21W (2.19 Å) presented here is closely similar to wild-type MexR. However, our extended analysis, by molecular dynamics and small-angle X-ray scattering, reveals that the mutation stabilizes a ground state that is deficient of DNA binding and is shared by both mutant and wild-type MexR, whereas the DNA-binding state is only transiently reached by the more flexible wild-type MexR. This population shift in the conformational ensemble is effected by mutation-induced allosteric coupling of contact networks that are independent in the wild-type protein. We propose that the MexR-R21W mutant mimics derepression by small-molecule binding to MarR proteins, and that the described allosteric model based on population shifts may also apply to other MarR family members.
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http://dx.doi.org/10.1016/j.str.2016.06.008DOI Listing
August 2016

Small-angle scattering and 3D structure interpretation.

Authors:
Jill Trewhella

Curr Opin Struct Biol 2016 10 30;40:1-7. Epub 2016 May 30.

The University of Sydney, New South Wales 2006, Australia. Electronic address:

This review focuses on advances in the application of solution small-angle scattering (SAS) in structural analysis of biomolecules and the complexes they form. Examples highlighted illustrate the unique contribution of SAS, using both X-rays and neutrons, in hybrid or integrative modelling methods. The increased information content when neutron scattering with contrast variation is used is a particular focus. Finally, progress toward an agreed reporting framework, the development of open data and model archives, and the importance of these initiatives is covered.
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http://dx.doi.org/10.1016/j.sbi.2016.05.003DOI Listing
October 2016

The outer-membrane export signal of Porphyromonas gingivalis type IX secretion system (T9SS) is a conserved C-terminal β-sandwich domain.

Sci Rep 2016 Mar 23;6:23123. Epub 2016 Mar 23.

Department of Microbiology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland.

In the recently characterized Type IX Secretion System (T9SS), the conserved C-terminal domain (CTD) in secreted proteins functions as an outer membrane translocation signal for export of virulence factors to the cell surface in the Gram-negative Bacteroidetes phylum. In the periodontal pathogen Porphyromonas gingivalis, the CTD is cleaved off by PorU sortase in a sequence-independent manner, and anionic lipopolysaccharide (A-LPS) is attached to many translocated proteins, thus anchoring them to the bacterial surface. Here, we solved the atomic structure of the CTD of gingipain B (RgpB) from P. gingivalis, alone and together with a preceding immunoglobulin-superfamily domain (IgSF). The CTD was found to possess a typical Ig-like fold encompassing seven antiparallel β-strands organized in two β-sheets, packed into a β-sandwich structure that can spontaneously dimerise through C-terminal strand swapping. Small angle X-ray scattering (SAXS) revealed no fixed orientation of the CTD with respect to the IgSF. By introducing insertion or substitution of residues within the inter-domain linker in the native protein, we were able to show that despite the region being unstructured, it nevertheless is resistant to general proteolysis. These data suggest structural motifs located in the two adjacent Ig-like domains dictate the processing of CTDs by the T9SS secretion pathway.
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http://dx.doi.org/10.1038/srep23123DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4804311PMC
March 2016

Structural Characterization of the Extracellular Domain of CASPR2 and Insights into Its Association with the Novel Ligand Contactin1.

J Biol Chem 2016 Mar 31;291(11):5788-5802. Epub 2015 Dec 31.

From the Child Health Institute of New Jersey and Departments of Neuroscience and Cell Biology and; Pediatrics, Robert Wood Johnson Medical School, Rutgers University, New Brunswick, New Jersey 08901,. Electronic address:

Contactin-associated protein-like 2 (CNTNAP2) encodes for CASPR2, a multidomain single transmembrane protein belonging to the neurexin superfamily that has been implicated in a broad range of human phenotypes including autism and language impairment. Using a combination of biophysical techniques, including small angle x-ray scattering, single particle electron microscopy, analytical ultracentrifugation, and bio-layer interferometry, we present novel structural and functional data that relate the architecture of the extracellular domain of CASPR2 to a previously unknown ligand, Contactin1 (CNTN1). Structurally, CASPR2 is highly glycosylated and has an overall compact architecture. Functionally, we show that CASPR2 associates with micromolar affinity with CNTN1 but, under the same conditions, it does not interact with any of the other members of the contactin family. Moreover, by using dissociated hippocampal neurons we show that microbeads loaded with CASPR2, but not with a deletion mutant, co-localize with transfected CNTN1, suggesting that CNTN1 is an endogenous ligand for CASPR2. These data provide novel insights into the structure and function of CASPR2, suggesting a complex role of CASPR2 in the nervous system.
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http://dx.doi.org/10.1074/jbc.M115.705681DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4786715PMC
March 2016

Clinically Linked Mutations in the Central Domains of Cardiac Myosin-Binding Protein C with Distinct Phenotypes Show Differential Structural Effects.

Structure 2016 Jan 10;24(1):105-115. Epub 2015 Dec 10.

School of Molecular Bioscience, The University of Sydney, NSW 2006, Australia. Electronic address:

The structural effects of three missense mutations clinically linked to hypertrophic cardiomyopathy (HCM) and located in the central domains of cardiac myosin-binding protein C (cMyBP-C) have been determined using small-angle scattering, infrared spectroscopy, and nuclear magnetic resonance spectroscopy. Bioinformatics and modeling were used to initially predict the expected structural impacts and assess the broader implications for function based on sequence conservation patterns. The experimental results generally affirm the predictions that two of the mutations (D745G, P873H) disrupt domain folding, while the third (R820Q) is likely to be entirely solvent exposed and thus more likely to have its impact through its interactions within the sarcomere. Each of the mutations is associated with distinct disease phenotypes, with respect to severity, stage of onset, and end phase. The results are discussed in terms of understanding key structural features of these domains essential for healthy function and the role they may play in disease development.
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http://dx.doi.org/10.1016/j.str.2015.11.001DOI Listing
January 2016

Structural basis of interprotein electron transfer in bacterial sulfite oxidation.

Elife 2015 Dec 19;4:e09066. Epub 2015 Dec 19.

Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Australia.

Interprotein electron transfer underpins the essential processes of life and relies on the formation of specific, yet transient protein-protein interactions. In biological systems, the detoxification of sulfite is catalyzed by the sulfite-oxidizing enzymes (SOEs), which interact with an electron acceptor for catalytic turnover. Here, we report the structural and functional analyses of the SOE SorT from Sinorhizobium meliloti and its cognate electron acceptor SorU. Kinetic and thermodynamic analyses of the SorT/SorU interaction show the complex is dynamic in solution, and that the proteins interact with Kd = 13.5 ± 0.8 μM. The crystal structures of the oxidized SorT and SorU, both in isolation and in complex, reveal the interface to be remarkably electrostatic, with an unusually large number of direct hydrogen bonding interactions. The assembly of the complex is accompanied by an adjustment in the structure of SorU, and conformational sampling provides a mechanism for dissociation of the SorT/SorU assembly.
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http://dx.doi.org/10.7554/eLife.09066DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4760952PMC
December 2015

Outcome of the First wwPDB Hybrid/Integrative Methods Task Force Workshop.

Structure 2015 Jul 18;23(7):1156-67. Epub 2015 Jun 18.

Research Collaboratory for Structural Bioinformatics Protein Data Bank, Center for Integrative Proteomics Research, Rutgers, The State University of New Jersey, Piscataway, NJ 08854, USA.

Structures of biomolecular systems are increasingly computed by integrative modeling that relies on varied types of experimental data and theoretical information. We describe here the proceedings and conclusions from the first wwPDB Hybrid/Integrative Methods Task Force Workshop held at the European Bioinformatics Institute in Hinxton, UK, on October 6 and 7, 2014. At the workshop, experts in various experimental fields of structural biology, experts in integrative modeling and visualization, and experts in data archiving addressed a series of questions central to the future of structural biology. How should integrative models be represented? How should the data and integrative models be validated? What data should be archived? How should the data and models be archived? What information should accompany the publication of integrative models?
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http://dx.doi.org/10.1016/j.str.2015.05.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4933300PMC
July 2015

The structure of human SFPQ reveals a coiled-coil mediated polymer essential for functional aggregation in gene regulation.

Nucleic Acids Res 2015 Apr 12;43(7):3826-40. Epub 2015 Mar 12.

School of Chemistry and Biochemistry, University of Western Australia, 35 Stirling Highway, Crawley, Western Australia 6009, Australia

SFPQ, (a.k.a. PSF), is a human tumor suppressor protein that regulates many important functions in the cell nucleus including coordination of long non-coding RNA molecules into nuclear bodies. Here we describe the first crystal structures of Splicing Factor Proline and Glutamine Rich (SFPQ), revealing structural similarity to the related PSPC1/NONO heterodimer and a strikingly extended structure (over 265 Å long) formed by an unusual anti-parallel coiled-coil that results in an infinite linear polymer of SFPQ dimers within the crystals. Small-angle X-ray scattering and transmission electron microscopy experiments show that polymerization is reversible in solution and can be templated by DNA. We demonstrate that the ability to polymerize is essential for the cellular functions of SFPQ: disruptive mutation of the coiled-coil interaction motif results in SFPQ mislocalization, reduced formation of nuclear bodies, abrogated molecular interactions and deficient transcriptional regulation. The coiled-coil interaction motif thus provides a molecular explanation for the functional aggregation of SFPQ that directs its role in regulating many aspects of cellular nucleic acid metabolism.
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http://dx.doi.org/10.1093/nar/gkv156DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4402515PMC
April 2015

Editorial overview--New frontiers of biophysical methods: tools for structural biology and beyond.

Curr Opin Struct Biol 2014 Oct 4;28:viii-x. Epub 2014 Oct 4.

Deputy Vice-Chancellor (Research), The University of Sydney, Room 646, Jane Foss Russell Building G02, Sydney, NSW 2006, Australia. Electronic address:

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http://dx.doi.org/10.1016/j.sbi.2014.09.005DOI Listing
October 2014

Report of the wwPDB Small-Angle Scattering Task Force: data requirements for biomolecular modeling and the PDB.

Structure 2013 Jun;21(6):875-81

School of Molecular Bioscience, The University of Sydney, NSW 2006, Australia.

This report presents the conclusions of the July 12-13, 2012 meeting of the Small-Angle Scattering Task Force of the worldwide Protein Data Bank (wwPDB; Berman et al., 2003) at Rutgers University in New Brunswick, New Jersey. The task force includes experts in small-angle scattering (SAS), crystallography, data archiving, and molecular modeling who met to consider questions regarding the contributions of SAS to modern structural biology. Recognizing there is a rapidly growing community of structural biology researchers acquiring and interpreting SAS data in terms of increasingly sophisticated molecular models, the task force recommends that (1) a global repository is needed that holds standard format X-ray and neutron SAS data that is searchable and freely accessible for download; (2) a standard dictionary is required for definitions of terms for data collection and for managing the SAS data repository; (3) options should be provided for including in the repository SAS-derived shape and atomistic models based on rigid-body refinement against SAS data along with specific information regarding the uniqueness and uncertainty of the model, and the protocol used to obtain it; (4) criteria need to be agreed upon for assessment of the quality of deposited SAS data and the accuracy of SAS-derived models, and the extent to which a given model fits the SAS data; (5) with the increasing diversity of structural biology data and models being generated, archiving options for models derived from diverse data will be required; and (6) thought leaders from the various structural biology disciplines should jointly define what to archive in the PDB and what complementary archives might be needed, taking into account both scientific needs and funding.
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http://dx.doi.org/10.1016/j.str.2013.04.020DOI Listing
June 2013

K7del is a common TPM2 gene mutation associated with nemaline myopathy and raised myofibre calcium sensitivity.

Brain 2013 Feb 31;136(Pt 2):494-507. Epub 2013 Jan 31.

Institute for Neuroscience and Muscle Research, Children’s Hospital at Westmead, Sydney, NSW 2145, Australia

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.
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http://dx.doi.org/10.1093/brain/aws348DOI Listing
February 2013

Effects of gadolinium chelate on the evolution of the nanoscale structure in peptide hydrogels.

Biopolymers 2012 23;98(1):50-8. Epub 2011 Jun 23.

Fischell Department of Bioengineering, University of Maryland, College Park, MD 20742.

Small-angle X-ray scattering (SAXS) was used to monitor peptide hydrogelation with and without the MRI contrast agent gadolinium chelate (Gd(III)-chelate). The gelators are a pair of decapeptides that are self-repulsive but mutually attractive. Gd(III)-chelate was either free or covalently bound to one of the decapeptides. Free and conjugated Gd(III)-chelate have the opposite effects on peptide gelation: free Gd(III)-chelate slows down gelation while having little effect on the cross-sectional area of peptide fibers; covalently conjugated Gd(III)-chelate speeds up gelation and results in peptide fibers with significantly greater cross-sectional area. After 24 h of gelation, the cross-sectional areas of hydrogels with no Gd(III)-chelate, with free Gd(III)-chelate and with conjugated Gd(III) chelate are 3700, 3800, and 6300 Å(2), respectively. Free Gd(III)-chelate is not incorporated into peptide fibers and remains in solution with little effect on MRI intensity upon gelation. In contrast, covalently conjugated Gd(III)-chelate is not only incorporated into peptide fibers, but further aggregates toward the center of the peptide fibers. In conclusion, to visualize hydrogelation using (1)H MRI, it is necessary to conjugate Gd(III)-chelate to the material covalently and use T(2)-weighted imaging.
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http://dx.doi.org/10.1002/bip.21689DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3559021PMC
April 2016

The role of heme binding by DNA-protective protein from starved cells (Dps) in the Tolerance of Porphyromonas gingivalis to heme toxicity.

J Biol Chem 2012 Dec 18;287(50):42243-58. Epub 2012 Oct 18.

University of Sydney, New South Wales 2006, Australia.

The widely expressed DNA-protective protein from starved-cells (Dps) family proteins are considered major contributors to prokaryotic resistance to stress. We show here that Porphyromonas gingivalis Dps (PgDps), previously described as an iron-storage and DNA-binding protein, also mediates heme sequestration. We determined that heme binds strongly to PgDps with an apparent K(d) of 3.7 × 10(-8) m and is coordinated by a single surface-located cysteine at the fifth axial ligand position. Heme and iron sequestered in separate sites by PgDps provide protection of DNA from H(2)O(2)-mediated free radical damage and were found to be important for growth of P. gingivalis under excess heme as the only iron source. Conservation of the heme-coordinating cysteine among Dps isoforms from the Bacteroidales order suggests that this function may be a common feature within these anaerobic bacteria.
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http://dx.doi.org/10.1074/jbc.M112.392787DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3516768PMC
December 2012

Calmodulin binds a highly extended HIV-1 MA protein that refolds upon its release.

Biophys J 2012 Aug;103(3):541-549

School of Molecular Bioscience, The University of Sydney, New South Wales, Australia. Electronic address:

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 Ca(2+) 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 Ca(2+)-signaling during viral processing.
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http://dx.doi.org/10.1016/j.bpj.2012.06.042DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3414894PMC
August 2012

Solution structure of the LIM-homeodomain transcription factor complex Lhx3/Ldb1 and the effects of a pituitary mutation on key Lhx3 interactions.

PLoS One 2012 25;7(7):e40719. Epub 2012 Jul 25.

School of Molecular Bioscience, University of Sydney, Sydney, New South Wales, Australia.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0040719PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3405102PMC
April 2013

The motif of human cardiac myosin-binding protein C is required for its Ca2+-dependent interaction with calmodulin.

J Biol Chem 2012 Sep 16;287(37):31596-607. Epub 2012 Jul 16.

School of Molecular Bioscience, Building G08, The University of Sydney, New South Wales 2006, Australia.

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 ΔS2. Here we report a novel Ca(2+)-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 Ca(2+)/CaM with a moderate affinity (K(D) ∼10 μM), which is similar to the affinity previously determined for myosin ΔS2. However, unlike the interaction with myosin ΔS2, the Ca(2+)/CaM interaction is unaffected by substitution with a triphosphorylated motif mimic. Further, Ca(2+)/CaM interacts with the highly conserved residues (Glu(319)-Lys(341)) toward the C-terminal end of the motif. Consistent with the Ca(2+) 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 Ca(2+) binding. Overall, Ca(2+)/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 Ca(2+) signaling pathways to help coordinate phosphorylation events and synchronize the multiple interactions between cMyBP-C, myosin, and actin during the heart muscle contraction.
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http://dx.doi.org/10.1074/jbc.M112.383299DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3438991PMC
September 2012

Publication guidelines for structural modelling of small-angle scattering data from biomolecules in solution.

Acta Crystallogr D Biol Crystallogr 2012 Jun 17;68(Pt 6):620-6. Epub 2012 May 17.

School of Molecular Bioscience, The University of Sydney, NSW 2006, Australia.

Small-angle scattering is becoming a mainstream technique for structural molecular biology. As such, it is important to establish guidelines for publication that will ensure that there is adequate reporting of the data and its treatment so that reviewers and readers can independently assess the quality of the data and the basis for any interpretations presented. This article presents a set of preliminary guidelines that emerged after consultation with the IUCr Commission on Small-Angle Scattering and other experts in the field and discusses the rationale for their application. At the 2011 Congress of the IUCr in Madrid, the Commission on Journals agreed to adopt these preliminary guidelines for the presentation of biomolecular structures from small-angle scattering data in IUCr publications. Here, these guidelines are outlined and the reasons for standardizing the way in which small-angle scattering data are presented.
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http://dx.doi.org/10.1107/S0907444912012073DOI Listing
June 2012

Reliable structural interpretation of small-angle scattering data from bio-molecules in solution--the importance of quality control and a standard reporting framework.

BMC Struct Biol 2012 May 17;12. Epub 2012 May 17.

School of Molecular Bioscience Building, University of Sydney, G08 Butlin Avenue, Sydney, NSW 2006, Australia.

Small-angle scattering is becoming an increasingly popular tool for the study of bio-molecular structures in solution. The large number of publications with 3D-structural models generated from small-angle solution scattering data has led to a growing consensus for the need to establish a standard reporting framework for their publication. The International Union of Crystallography recently established a set of guidelines for the necessary information required for the publication of such structural models. Here we describe the rationale for these guidelines and the importance of standardising the way in which small-angle scattering data from bio-molecules and associated structural interpretations are reported.
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http://dx.doi.org/10.1186/1472-6807-12-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3412165PMC
May 2012