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Benjamin T. Porebski, Adrian A. Nickson, David E. Hoke, Morag R. Hunter, Liguang Zhu, Sheena McGowan, Geoffrey I. Webb and Ashley M. Buckle
Protein Engineering, Design and Selection (2015) 28 (3): 67-78. doi: 10.1093/protein/gzv002
Consensus protein design is a rapid and reliable technique for the improvement of protein stability, which relies on the use of homologous protein sequences. To enhance the stability of a fibronectin type III (FN3) domain, consensus design was employed using an alignment of 2123 sequences. The resulting FN3 domain, FN3con, has unprecedented stability, with a melting temperature >100°C, a ΔGD−N of 15.5 kcal mol−1 and a greatly reduced unfolding rate compared with wild-type. To determine the underlying molecular basis for stability, an X-ray crystal structure of FN3con was determined to 2.0 Å and compared with other FN3 domains of varying stabilities. The structure of FN3con reveals significantly increased salt bridge interactions that are cooperatively networked, and a highly optimized hydrophobic core. Molecular dynamics simulations of FN3con and comparison structures show the cooperative power of electrostatic and hydrophobic networks in improving FN3con stability. Taken together, our data reveal that FN3con stability does not result from a single mechanism, but rather the combination of several features and the removal of non-conserved, unfavorable interactions. The large number of sequences employed in this study has most likely enhanced the robustness of the consensus design, which is now possible due to the increased sequence availability in the post-genomic era. These studies increase our knowledge of the molecular mechanisms that govern stability and demonstrate the rising potential for enhancing stability via the consensus method.
Understanding the structural dynamics of TCR-pMHC complex interactions.
Kass I, Buckle AM, Borg NA.
Trends Immunol. 2014 Nov 11;35(12):604-612. doi: 10.1016/j.it.2014.10.005. [Epub ahead of print]
Exponential growth in simulation complexity over time. Growth is driven by the doubling of computer power every 24 months coupled with algorithmic advances. The complexity of the simulation is represented by the product of the timescale and system size, on the y-axis. Filled circles represent published molecular dynamics (MD) studies.
Dynamics plays an important but underappreciated role in the interaction between the T cell receptor (TCR) and peptide-bound major histocompatibility complex (pMHC). Crystallographic studies have provided key molecular insights into this interaction; however, due to inherent features of the structural approach, the image of TCR-pMHC interactions that has emerged is a static one. In this review, we discuss how molecular dynamics (MD) simulations can complement and extend current experimental methods aimed at examining TCR-pMHC dynamics. We review the insights obtained from studies that leverage MD approaches, and propose that an integrative strategy that harnesses both MD simulations and structural and biophysical methods will provide new inroads into understanding the transitory and dynamic molecular events that dictate TCR signaling and T cell activation.
Meyer GR, Aragão D, Mudie NJ, Caradoc-Davies TT, McGowan S, Bertling PJ, Groenewegen D, Quenette SM, Bond CS, Buckle AM, Androulakis S.
Acta Crystallogr D Biol Crystallogr. 2014 Oct;70(Pt 10):2510-9. doi: 10.1107/S1399004714016174. Epub 2014 Sep 30.
Samson AL, Knaupp AS, Kass I, Kleifeld O, Marijanovic EM, Hughes VA, Lupton CJ, Buckle AM, Bottomley SP, Medcalf RL.
J Biol Chem. 2014 Sep 26;289(39):26922-36. doi: 10.1074/jbc.M114.570275. Epub 2014 Aug 1.
Arjomand A, Baker MA, Li C, Buckle AM, Jans DA, Loveland KL, Miyamoto Y.
FASEB J. 2014 Aug;28(8):3480-93. doi: 10.1096/fj.13-244913. Epub 2014 Apr 30.
Cofactor-dependent conformational heterogeneity of GAD65 and its role in autoimmunity and neurotransmitter homeostasis
See also Monash Media releases:
The human neuroendocrine enzyme glutamate decarboxylase (GAD) catalyses the synthesis of the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) using pyridoxal 5′-phosphate as a cofactor. GAD exists as two isoforms named according to their respective molecular weights: GAD65 and GAD67. Although cytosolic GAD67 is typically saturated with the cofactor (holoGAD67) and constitutively active to produce basal levels of GABA, the membrane-associated GAD65 exists mainly as the inactive apo form. GAD65, but not GAD67, is a prevalent autoantigen, with autoantibodies to GAD65 being detected at high frequency in patients with autoimmune (type 1) diabetes and certain other autoimmune disorders. The significance of GAD65 autoinactivation into the apo form for regulation of neurotransmitter levels and autoantibody reactivity is not understood. We have used computational and experimental approaches to decipher the nature of the holo → apo conversion in GAD65 and thus, its mechanism of autoinactivation. Molecular dynamics simulations of GAD65 reveal coupling between the C-terminal domain, catalytic loop, and pyridoxal 5′-phosphate–binding domain that drives structural rearrangement, dimer opening, and autoinactivation, consistent with limited proteolysis fragmentation patterns. Together with small-angle X-ray scattering and fluorescence spectroscopy data, our findings are consistent with apoGAD65 existing as an ensemble of conformations. Antibody-binding kinetics suggest a mechanism of mutually induced conformational changes, implicating the flexibility of apoGAD65 in its autoantigenicity. Although conformational diversity may provide a mechanism for cofactor-controlled regulation of neurotransmitter biosynthesis, it may also come at a cost of insufficient development of immune self-tolerance that favors the production of GAD65 autoantibodies.
We have developed two software plugins for the Mac OSX operating system that allow rapid and convenient visualization of PDB files and X-ray diffraction images directly within the file browser, without the need of full-featured applications. We have just published a paper describing this work:
Shown below are views of a folder containing PDB files. Shown on the right is a Finder window of a folder containing PDB files with the PyMol rendered cartoon thumbnail. On the left is a QuickLook popup of the same PDB file using the Jolecule viewer.
Hargreaves CE, Grasso M, Hampe CS, Stenkova A, Atkinson S, Joshua GW, Wren BW, Buckle AM, Dunn-Walters D, Banga JP.
Wijeyewickrema LC, Yongqing T, Tran TP, Thompson PE, Viljoen JE, Coetzer TH, Duncan RC, Kass I, Buckle AM, Pike RN.
de Veer SJ, Ukolova SS, Munro CA, Swedberg JE, Buckle AM, Harris JM.
Biopolymers. 2013 Mar 11. doi: 10.1002/peps.22231. [Epub ahead of print]
Langendorf CG, Tuck KL, Key TL, Fenalti G, Pike RN, Rosado CJ, Wong AS, Buckle AM, Law RH, Whisstock JC.
Biosci Rep. 2013 Jan 11;33(1):137-44. doi: 10.1042/BSR20120111.
After 6 years of work!!!!
McGowan S, Buckle AM, Mitchell MS, Hoopes JT, Gallagher DT, Heselpoth RD, Shen Y, Reboul CF, Law RH, Fischetti VA, Whisstock JC, Nelson DC. (2012) X-ray crystal structure of the streptococcal specific phage lysin PlyC. Proc Natl Acad Sci U S A. 2012 Jul 17.
Reboul CF, Meyer GR, Porebski BT, Borg NA, Buckle AM (2012) Epitope Flexibility and Dynamic Footprint Revealed by Molecular Dynamics of a pMHC-TCR Complex. PLoS Comput Biol 8(3): e1002404. doi:10.1371/journal.pcbi.1002404
Reboul CF, Porebski BT, Griffin MDW, Dobson RCJ, Perugini MA, et al. (2012) Structural and Dynamic Requirements for Optimal Activity of the Essential Bacterial Enzyme Dihydrodipicolinate Synthase. PLoS Comput Biol 8(6): e1002537. doi:10.1371/journal.pcbi.1002537
Atkinson SC, Dogovski C, Downton MT, Pearce FG, Reboul CF, Buckle AM, Gerrard JA, Dobson RC, Wagner J, Perugini MA.et al. (2012) Crystal, Solution andIn silico Structural Studies of Dihydrodipicolinate Synthase from the Common Grapevine. PLoS ONE 7(6): e38318. doi:10.1371/journal.pone.0038318
The Rate of PolyQ-Mediated Aggregation Is Dramatically Affected by the Number and Location of Surrounding Domains
Amy L. Robertson, Mark A. Bate, Ashley M. Buckle, Stephen P. Bottomley
Computational methods for studying serpin conformational change and structural plasticity.
Kass I, Reboul CF, Buckle AM.
Refolding Your Protein with a Little Help from REFOLD
Jennifer Phan, Nasrin Yamout, Jason Schmidberger, Stephen P. Bottomley and Ashley M. Buckle
PROTEIN FOLDING, MISFOLDING, AND DISEASE
Methods in Molecular Biology, 2011, Volume 752, 45-57, DOI: 10.1007/978-1-60327-223-0_4
Characterisation of Peptide Microarrays for Studying Antibody-Antigen Binding Using Surface Plasmon Resonance Imagery. PLoS ONE 5(8): e12152. doi:10.1371/journal.pone.0012152
Claude Nogues, Hervé Leh, Christopher G. Langendorf, Ruby H. P. Law, Ashley M. Buckle#*, Malcolm Buckle#*
1 Dynamics of Macromolecular Complexes, Laboratoire de Biologie et Pharmacologie Appliquée, UMR 8113 du CNRS, Institut d’Alembert, Ecole Normale Supérieure de Cachan, Cachan, France
Non-specific binding to biosensor surfaces is a major obstacle to quantitative analysis of selective retention of analytes at immobilized target molecules. Although a range of chemical antifouling monolayers has been developed to address this problem, many macromolecular interactions still remain refractory to analysis due to the prevalent high degree of non-specific binding. We describe how we use the dynamic process of the formation of self assembling monolayers and optimise physical and chemical properties thus reducing considerably non-specific binding and allowing analysis of specific binding of analytes to immobilized target molecules.
We illustrate this approach by the production of specific protein arrays for the analysis of interactions between the 65kDa isoform of human glutamate decarboxylase (GAD65) and a human monoclonal antibody. Our data illustrate that we have effectively eliminated non-specific interactions with the surface containing the immobilised GAD65 molecules. The findings have several implications. First, this approach obviates the dubious process of background subtraction and gives access to more accurate kinetic and equilibrium values that are no longer contaminated by multiphase non-specific binding. Second, an enhanced signal to noise ratio increases not only the sensitivity but also confidence in the use of SPR to generate kinetic constants that may then be inserted into van’t Hoff type analyses to provide comparative ΔG, ΔS and ΔH values, making this an efficient, rapid and competitive alternative to ITC measurements used in drug and macromolecular-interaction mechanistic studies. Third, the accuracy of the measurements allows the application of more intricate interaction models than simple Langmuir monophasic binding.
The detection and measurement of antibody binding by the type 1 diabetes autoantigen GAD65 represents an example of an antibody-antigen interaction where good structural, mechanistic and immunological data are available. Using SPRi we were able to characterise the kinetics of the interaction in greater detail than ELISA/RIA methods. Furthermore, our data indicate that SPRi is well suited to a multiplexed immunoassay using GAD65 proteins, and may be applicable to other biomarkers.
Interview about our TARDIS project:
Monash University have written a piece about TARDIS in their ‘Monash Memo‘
Schmidberger JS, Bate MA, Reboul CF, Androulakis SG, Phan JMN, Whisstock JC, Goscinski WJ, Abramson A, and Buckle AM (2010) MrGrid: A Portable Grid Based Molecular Replacement Pipeline. PLoS One. Apr 6;5(4):e10049. PubMed link
Apple University Consortium (AUC) in Australia wrote an article on our grid computing in their newsletter Wheels of the Mind (PDF)
Konagurthu AS, Reboul CF, Schmidberger JS, Irving, JA, Lesk AM, Stuckey PJ, Whisstock JC, and Buckle AM (2010) MUSTANG-MR Structural Sieving Server: Applications in Protein Structural Analysis and Crystallography. PLoS One. Apr 6;5(4):e10048. PubMed link