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

PEDS front small


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.

New papers published

Operation of the Australian Store.Synchrotron for macromolecular crystallography.

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.

Oxidation of an exposed methionine instigates the aggregation of glyceraldehyde-3-phosphate dehydrogenase.

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.

The α-importome of mammalian germ cell maturation provides novel insights for importin biology.

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.

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.


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.