Connecting Unexplored Protein Crystal Structures to Enzymatic Function

OBJECTIVES/GOALS: Neoantigen vaccine immunotherapies have shown promise in clinical trials, but identifying which peptides to include in a vaccine remains a challenge. We aim to establish that molecular structural features can help predict which neoantigens to target to achieve tumor regression. METHODS/STUDY POPULATION: Proteins were prepared by recombinant expression in E. coli followed by in vitro refolding. Correctly folded proteins were purified by chromatography. Affinities of protein-protein interactions were measured by surface plasmon resonance (SPR) and thermal stabilities of proteins were determined by differential scanning fluorimetry. All experiments were performed at least in triplicate. Protein crystals were obtained by hanging drop vapor diffusion. The protein crystal structures were solved by molecular replacement and underwent several rounds of automated refinement. Molecular dynamics simulations were performed using the AMBER molecular dynamics package. RESULTS/ANTICIPATED RESULTS: A T cell receptor (TCR) expressed by tumor-infiltrating T cells exhibited a 20-fold stronger binding affinity to the neoantigen peptide compared to the self-peptide. X-ray crystal structures of the peptides with the major histocompatibility complex (MHC) protein demonstrated that a non-mutated residue in the peptide samples different positions with the mutation. The difference in conformations of the non-mutated residue was supported by molecular dynamics simulations. Crystal structures of the TCR engaging both peptide/MHCs suggested that the conformation favored by the mutant peptide was crucial for TCR binding. The TCR bound the neoantigen/MHC with faster binding kinetics. DISCUSSION/SIGNIFICANCE OF IMPACT: Our results suggest that the mutation impacts the conformation of another residue in the peptide, and this alteration allows for more favorable T cell receptor binding to the neoantigen. This highlights the potential of non-mutated residues in contributing to neoantigen recognition.

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Ensemble Refinement of Protein Crystal Structures: Validation and Application

Structure ◽

10.1016/j.str.2007.06.019 ◽

2007 ◽

Vol 15 (9) ◽

pp. 1040-1052 ◽

Cited By ~ 131

Author(s):

Elena J. Levin ◽

Dmitry A. Kondrashov ◽

Gary E. Wesenberg ◽

George N. Phillips

Keyword(s):

Crystal Structures ◽

Protein Crystal

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T-to-R switch of muscle fructose-1,6-bisphosphatase involves fundamental changes of secondary and quaternary structure

Acta Crystallographica Section D Structural Biology ◽

10.1107/s2059798316001765 ◽

2016 ◽

Vol 72 (4) ◽

pp. 536-550 ◽

Cited By ~ 15

Author(s):

Jakub Barciszewski ◽

Janusz Wisniewski ◽

Robert Kolodziejczyk ◽

Mariusz Jaskolski ◽

Dariusz Rakus ◽

...

Keyword(s):

Crystal Structures ◽

Catalytic Mechanism ◽

Quaternary Structure ◽

Homo Sapiens ◽

Helical Structure ◽

Smooth Transition ◽

Muscle Enzyme ◽

Enzymatic Function ◽

Fructose 1,6 Bisphosphatase ◽

Key Enzyme

Fructose-1,6-bisphosphatase (FBPase) catalyzes the hydrolysis of fructose 1,6-bisphosphate to fructose 6-phosphate and is a key enzyme of gluconeogenesis and glyconeogenesis and, more generally, of the control of energy metabolism and glucose homeostasis. Vertebrates, and notablyHomo sapiens, express two FBPase isoforms. The liver isozyme is expressed mainly in gluconeogenic organs, where it functions as a regulator of glucose synthesis. The muscle isoform is expressed in all cells, and recent studies have demonstrated that its role goes far beyond the enzymatic function, as it can interact with various nuclear and mitochondrial proteins. Even in its enzymatic function, the muscle enzyme is different from the liver isoform, as it is 100-fold more susceptible to allosteric inhibition by AMP and this effect can be abrogated by complex formation with aldolase. All FBPases are homotetramers composed of two intimate dimers: the upper dimer and the lower dimer. They oscillate between two conformational states: the inactive T form when in complex with AMP, and the active R form. Parenthetically, it is noted that bacterial FBPases behave somewhat differently, and in the absence of allosteric activators exist in a tetramer–dimer equilibrium even at relatively high concentrations. [Hineset al.(2007),J. Biol. Chem.282, 11696–11704]. The T-to-R transition is correlated with the conformation of the key loop L2, which in the T form becomes `disengaged' and unable to participate in the catalytic mechanism. The T states of both isoforms are very similar, with a small twist of the upper dimer relative to the lower dimer. It is shown that at variance with the well studied R form of the liver enzyme, which is flat, the R form of the muscle enzyme is diametrically different, with a perpendicular orientation of the upper and lower dimers. The crystal structure of the muscle-isozyme R form shows that in this arrangement of the tetramer completely new protein surfaces are exposed that are most likely targets for the interactions with various cellular and enzymatic partners. The cruciform R structure is stabilized by a novel `leucine lock', which prevents the key residue, Asp187, from locking loop L2 in the disengaged conformation. In addition, the crystal structures of muscle FBPase in the T conformation with and without AMP strongly suggest that the T-to-R transition is a discrete jump rather than a shift of an equilibrium smooth transition through multiple intermediate states. Finally, using snapshots from three crystal structures of human muscle FBPase, it is conclusively demonstrated that the AMP-binding event is correlated with a β→α transition at the N-terminus of the protein and with the formation of a new helical structure.

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Automated Real-Space Refinement of Protein Crystal Structures Using a Realistic Backbone Move Set

Biophysical Journal ◽

10.1016/j.bpj.2010.12.1903 ◽

2011 ◽

Vol 100 (3) ◽

pp. 312a ◽

Cited By ~ 1

Author(s):

Esmael J. Haddadian ◽

Haipeng Gong ◽

Abhishek K. Jha ◽

Xiaojing Yang ◽

Joe DeBartolo ◽

...

Keyword(s):

Crystal Structures ◽

Real Space ◽

Protein Crystal

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Structure determination from powder diffraction data

Acta Crystallographica Section A Foundations of Crystallography ◽

10.1107/s0108767307064252 ◽

2007 ◽

Vol 64 (1) ◽

pp. 52-64 ◽

Cited By ~ 174

Author(s):

W. I. F. David ◽

K. Shankland

Keyword(s):

Crystal Structures ◽

Powder Diffraction ◽

Diffraction Data ◽

Structure Determination ◽

Optimization Methods ◽

Protein Crystal ◽

Powder Diffraction Data ◽

Structure Solution ◽

Combined Use ◽

Complementary Techniques

Advances made over the past decade in structure determination from powder diffraction data are reviewed with particular emphasis on algorithmic developments and the successes and limitations of the technique. While global optimization methods have been successful in the solution of molecular crystal structures, new methods are required to make the solution of inorganic crystal structures more routine. The use of complementary techniques such as NMR to assist structure solution is discussed and the potential for the combined use of X-ray and neutron diffraction data for structure verification is explored. Structures that have proved difficult to solve from powder diffraction data are reviewed and the limitations of structure determination from powder diffraction data are discussed. Furthermore, the prospects of solving small protein crystal structures over the next decade are assessed.

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