New coNformatioNal properties of sH 2 domaiN biNdiNg pocket

the conformational changes of proteins play an important role in biological functioning such as ligand-protein and protein-protein interactions. the aim of the work was to investigate the conformational movement of most represented SH2 domains. It was found that SH2 domain binding pocket includes both flexible and not flexible regions: the central area of the binding pocket is the most unflexible, whereas the pTyr-binding and hydrophobic zones are the most flexible. Results of the computer analysis revealed new conformational properties of SH2 domain, which are important for drug design.


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t is known that proteins undergo conformational changes in the course of their functioning.Conformations, responsible for a particular property, may not correspond to the global minimum, and in some cases the ensemble of conformations can be responsible for an observed behavior.So, the conformation with minimal energy is not enough for representing the molecule.Conformational changes may range from small local movements to large domain motions, which affect the protein functions.Typical example is the transformation of enzyme active site (close, open) that provides chemical modification of ligand (increasing selectivity to active site of enzyme).That is why conformational analysis of protein plays an important role in the field of bioinformatics for protein secondary structure prediction and drug design.
It is known that Src homology 2 (SH2) domains are small protein modules consisting of approximately 100 amino acids which form two α-helix and seven β-sheets.Human genome encodes about 120 SH2 domains which are included in 110 different proteins, such as protein kinases (Src, Lck), phosphatases (SHP2, SHIP2), phospholipases (PLC1), transcription factors (STAT), regulatory proteins (SOCS), adapter proteins (Grb2), structural proteins (SHC) and others.These domains play an important role in intracellular signaling in complex with different protein, mainly Src oncoprotein [1].Binding pocket of SH2 domain is characterized by a well-defined placement of pTyr-binding part (usually ArgB5 creates a strong hydrogen bond with phosphotyrosine), a central part (hydrogen bond between NH group of ligand and O of His) and a large hydrophobic part (interacts with a hydrophobic part of ligand) [2][3][4].The main function of SH2 domain is pTyr-binding recognition [5].Recent research has shown that each SH2 domain binds only to specific phosphotyrosine-containing fragments (e.g. the Src SH2 domain recognizes GluGlu-Ile (binding fragment pYEEI), Grb2 SH2 domain binds to pYVNV) [6].Based on this, it is possible to classify SH2 domains according to the specificity of recognition of pTyr residue with C-terminus [6].
In the work we analyzed different possible conformations of SH2 domains.For generation of the conformations the LowModeMD Search method was used.It employs a short ~1 ps run of Molecular Dynamics (MD) at constant temperature followed by all-atom energy minimization [7].
The aim of the work was to define the main flexible and rigid regions of SH2 domain.However, we must note that complete understanding of this effect requires detailed study of thermodynamic proper ties of SH2 domains.
Search for new conformations of SH2 domains was performed by using MOE software package [8] where Low Mode Search (а short molecular dynamics simulation using velocities with small kinetic energy on the high-frequency vibrational modes) method was applied.So, the conformation of SH2 domain was generated according to the following options: -Rejection Limit (the numbers of contiguous attempts which must be made to generate new conformation prior to terminating the search) -100; -Iteration Limit (maximum number of attempts to generate new conformation (independent of the uniqueness of each generated conformation)) -10; -RMS Gradient (if root mean square gradient falls below the specified value the energy minimization is terminated) -0.005; -MM Iteration Limit (the maximum number of energy minimization steps performed for each conformer energy minimization) -50; -RMSD Limit (if two conformations have a Rmsd less than the specified value, they are considered indistinguishable) -0.25 Å, 0.5 Å and 0.75 Å; -Strain Cutoff (energy minimized conformations will be discarded if their potential energy is greater than E min +s, where E min is the lowest energy among the minimized conformations and s is the value specified for Strain Cutoff (in kcal/mol)) -7.
The obtained conformations were energy minimized in Cartesian coordinates system.

result and discussion
The mobilities of eight most representative SH2 domains were determined.The example of SH2 domain conformation superposition is presented in Fig. 1.RMSD (root mean square deviations) for all obtained conformations are given in Table and for all residues are presented in Fig 2 .Generally, 39 (from four to six per structure) conformations were obtained.The observed average RMSD of any conformation varies between 1 Å and 3.5 Å, and only in one case it is equal to 5.17 Å (conformation N3 of PDB ID 1O49).To take into consideration that "DEloop, βE -sheet, EF-loop and FB-loop" region is located only in one part of domain, we examined them together in Fig. 1.
The central part of the pocket is the most stable part.Only in two cases (2FCI -2.3 Å and 2GE9 -2.5 Å) RMSD is greater than 2 Å.In all other situations RMSD is less (1O49 -1.3 Å, 1UUS -1 Å, 2JYQ -1.8 Å, 2K7A -2 Å, 2KK6 -1.8 Å and 3IN7 -1.6 Å).Almost in all cases βD-sheet and αAhelix create that part.But there are few exceptions.So, in 1UUS in the creation of this part of pocket BC-sheet (TYR 625) and "DE-loop, βE-sheet, EFloop and FB-loop" part (VAL 639 and GLN 640) participate too; in 2FCI -CD-loop and in 2K7A -BC-sheet and "DE-loop, βE-sheet, EF-loop and FBloop".експериментальні роботи It was found that αA-helix, BC-loop, CD-loop, αB-helix, ВG-loop and "DE-loop, βE-sheet, EF-loop and FB-loop" secondary structures demonstrate essential contribution to the total variability of SH2 domain conformations.In opposite to this, the flexibility of AA-loop, AB-loop, βB-sheet, βC-sheet, βD-sheet secondary structures are lower.The most flexible conformation was found in PDB file 1O49, namely, conformation N3 (RMSD limit 0.25 Å).The binding pocket is less flexible than other parts of the domain.The hydrophobic and pTyr-binding parts are the most flexible in the binding pocket, but the central part is rigid.