Protein Engineering of FN3 published

Structural and dynamic properties that govern the stability of an engineered fibronectin type III domain

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

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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.

Review Published on T-cell Receptor Dynamics

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/ [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.

GAD65 conformational dynamics paper published in PNAS

Cofactor-dependent conformational heterogeneity of GAD65 and its role in autoimmunity and neurotransmitter homeostasis

Kass et al, PNAS, Early online edition June 9th, 2014

See also Monash Media releases:

‘Jekyll and Hyde’ protein linked to type 1 diabetes

The autoimmune cost of shape-shifting brain protein 

Open and closed dimers of GAD65 and the mechanism of dimer opening in holo → apo conversion

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.


Interactive visualization tools for the structural biologist

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:

Porebski BT, Ho BK, Buckle AM. (2013) Interactive visualization tools for the structural biologist. J. Appl. Cryst. (2013). 46, 1518-1520.

Project page and download

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.

New papers published

Yersinia enterocolitica Provides the Link between Thyroid-Stimulating Antibodies and Their Germline Counterparts in Graves’ Disease.

Hargreaves CE, Grasso M, Hampe CS, Stenkova A, Atkinson S, Joshua GW, Wren BW, Buckle AM, Dunn-Walters D, Banga JP.

Molecular determinants of the substrate specificity of the complement initiating protease, C1r.

Wijeyewickrema LC, Yongqing T, Tran TP, Thompson PE, Viljoen JE, Coetzer TH, Duncan RC, Kass I, Buckle AM, Pike RN.

J Biol Chem. 2013 Apr 15. [Epub ahead of print]

Mechanism-based selection of a potent kallikrein-related peptidase 7 inhibitor from a versatile library based on the sunflower trypsin inhibitor SFTI-1.

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]

Structural characterization of the mechanism through which human glutamic acid decarboxylase auto-activates.

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.

6 New MD papers published

Peptide dynamics in the MHC groove


Bosco K Ho, David Perahia, Ashley M Buckle (2012)  Hybrid approaches to molecular simulation.  Current Opinion in Structural Biology, Volume 22, Issue 3, Pages 386-393.  


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


Kass, Knaupp, Bottomley, Buckle. (2012). Conformational properties of the disease-causing Z variant of alpha1 antitrypsin revealed by molecular dynamics simulations.    Biophys. J.102(12):2856-65.


Atkinson SC, Dogovski C, Downton MT, Pearce FG, Reboul CF, Buckle AM, Gerrard JA, Dobson RC, Wagner J, Perugini 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


Kass I, Reboul CF, Buckle AM. (2012) Computational methods for studying serpin conformational change and structural plasticity. Methods Enzymol. 2011;501:295-323.

2 New Papers

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

J Mol Biol. 2011 Nov 4;413(4):879-87. Epub 2011 Sep 16.

Computational methods for studying serpin conformational change and structural plasticity.

Kass I, Reboul CF, Buckle AM.

Methods Enzymol. 2011;501:295-323.

New Paper Published!

PolyQ: a database describing the sequence and domain context of polyglutamine repeats in proteins

Amy L. Robertson, Mark A. Bate, Steve G. Androulakis, Stephen P. Bottomley, and Ashley M. Buckle

Nucl. Acids Res. (2010) doi: 10.1093/nar/gkq1100
First published online: November 8, 2010
This article is Open Access

Download the PDF

The polyglutamine diseases are caused in part by a gain-of-function mechanism of neuronal toxicity involving protein conformational changes that result in the formation and deposition of β-sheet rich aggregates. Recent evidence suggests that the misfolding mechanism is context-dependent, and that properties of the host protein, including the domain architecture and location of the repeat tract, can modulate aggregation. In order to allow the bioinformatic investigation of the context of polyglutamines, we have constructed a database, PolyQ.   We have collected the sequences of all human proteins containing runs of seven or more glutamine residues and annotated their sequences with domain information. PolyQ can be interrogated such that the sequence context of polyglutamine repeats in disease and non-disease associated proteins can be investigated.

New paper published

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

2 Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia

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.

Methodology/Principal Findings
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.

Two new papers published


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

Four new papers published:

Swedberg JE, Nigon LV, Reid JC, de Veer SJ, Walpole CM, Stephens CR, Walsh TP, Takayama TK, Hooper JD, Clements JA, Buckle AM & Harris, JM.  (2009) Substrate-guided design of a potent and selective kallikrein-related peptidase inhibitor for kallikrein 4.  Chemistry & Biology, 16, 633-43. PubMed Link

Fischer K,Langendorf  CG, Irving  JA, Reynolds S, Willis C, Beckham S, Law RHP, Yang S, Bashtannyk-Puhalovich TA, McGowan S, Whisstock JC, PikeRN, KempDJ, and Buckle AM  (2009) Structural Mechanisms of Inactivation in Scabies Mite Serine Protease Paralogues. J. Mol. Biol. 390(4):635-45. PubMed Link

Fenalti G and Buckle AM (2010) Structural Biology of the GAD Autoantigen. Autoimmunity Reviews 9(3):148-52. Epub 2009 May 22. PubMed Link

Arafat Y, Fenalti G, Whisstock JC, Mackay IR, Garcia de la Banda M, Rowley MJ and Buckle AM (2009) Structural Determinants of GAD Antigenicity. Molecular Immunology, 47(2-3):493-505. PubMed Link

TARDIS article on P212121

The crystallography blog P212121 have posted an article on TARDIS  entitled “TARDIS for the Storage of X-ray Diffraction Images“.

This comes as a follow-up to their recent poll “Do we need an X-ray Diffraction Image Data Bank?“.

The poll is still open, but currently 81% of P212121 readers are voting for that the crystallography community does need an open, freely available diffraction image data bank.

Be sure to head over to P212121 and have your say.