SH2db, an information system for the SH2 domain

Abstract

SH2 domains are key mediators of phosphotyrosine-based signalling, and therapeutic targets for diverse, mostly oncological, disease indications. They have a highly conserved structure with a central beta sheet that divides the binding surface of the protein into two main pockets, responsible for phosphotyrosine binding (pY pocket) and substrate specificity (pY + 3 pocket). In recent years, structural databases have proven to be invaluable resources for the drug discovery community, as they contain highly relevant and up-to-date information on important protein classes. Here, we present SH2db, a comprehensive structural database and webserver for SH2 domain structures. To organize these protein structures efficiently, we introduce (i) a generic residue numbering scheme to enhance the comparability of different SH2 domains, (ii) a structure-based multiple sequence alignment of all 120 human wild-type SH2 domain sequences and their PDB and AlphaFold structures. The aligned sequences and structures can be searched, browsed and downloaded from the online interface of SH2db (http://sh2db.ttk.hu), with functions to conveniently prepare multiple structures into a Pymol session, and to export simple charts on the contents of the database. Our hope is that SH2db can assist researchers in their day-to-day work by becoming a one-stop shop for SH2 domain related research.

Graphical Abstract

Figure 1.

Schematic representation of the structurally conserved segments of SH2 domains. Grey represents the terminals, black disordered loops, blue turns between strands, turquoise beta strands and purple alpha helices. Generic numbered positions are assigned to all beta strands and alpha helices. (A) In the most common segment layout of SH2 domains, strand bD is followed by two shorter strands bE and bF, then the domain ends with the aB helix, with all of them connected by loops or turns. (B) In the STAT proteins, the bE and bF strands with their flanking loops/turns are missing and instead the aB’ helix is present, which connects to bD and aB without any loops or turns.

Figure 2.

Phylogenetic tree of the SH2 domain containing proteins using the sequence alignment of positions with a generic number. The ‘-C’ tag denotes the C-terminal SH2 domain (in proteins with two SH2 domains). The tree was made with Biopython's Phylo module and iTOL (60).

Figure 3.

SH2db provides two main ways for accessing the underlying database. (A) From the Browse page, the user can hierarchically navigate first to entries of specific proteins, and then to specific structures. The entries contain external database links, a sequence viewer, download options and an interactive NGLviewer panel for quick visualization. (B) The Search page provides functionalities to filter the underlying database via an interactive sequence viewer and download arbitrary selections of sequences or structures. The toggle button (1) switches between including structure entries or restricting the table to canonical protein sequences extracted from UniProt. The Domain column (2) lists the PDB IDs or marks the UniProt sequences and AlphaFold models by their Uniprot ID, followed by ‘N’ or ‘C’ for proteins with dual SH2 domains (or ‘N’ by default for single-SH2 proteins). The table can be filtered by any combination of fields, including individual amino acid positions (3). Selections can be downloaded as sequences (fasta), structures (pdb) or fed into a backend script to generate a Pymol session (4), which shows the selected structures superposed, and the selected residues highlighted as sticks, for a quick and easy structure comparison.

Figure 4.

Structures (left) of the wild-type (A, PDB: 6MBW), N642H ‘tight-bD’ (B, PDB: 6MBZ, chain B) and ‘loose-bD’ (C, PDB: 6MBZ, chain A) conformation STAT5B SH2 domains (49), and the docking poses of the GpYLVLDKW peptide (green) against these domains (right). In the ‘loose-bD’ conformation, the dissociated bD strand contributes to the formation of a small subpocket that can accommodate the N-terminal end of the phospho-peptide within the pY pocket.

Figure 5.

(A) Excerpt from the multiple sequence viewer on the Search page: the D61G mutant quickly stands out from the large number of available SHP2 structures. (B) Structural requirements for SH2-PTP binding (PDB structure 6BMW (59)): in the closed conformation, the D61 sidechain of the N-SH2 domain (red) bDbE loop can establish a salt bridge with the R465 residue of the PTP domain (white), allowing the N-SH2 domain to block access to the so-called ‘phosphate cradle’ of the active site (cyan). The Y62^bE.48 sidechain was proposed to interact with the hydrophobic part of the pocket; alternatively, if deprotonated, it could form an additional salt bridge with the proximal cluster of positively charged residues of the PTP domain (highlighted as sticks). (C) Compared to the wild-type N-SH2 domain (red), the oncogenic D61G mutant (blue, PDB structure 4H1O, https://www.rcsb.org/structure/4H1O) misses the crucial negatively charged sidechain and is thus not able to form the anchoring salt bridge, resulting in a loss of auto-regulation (permanently active state). The C-SH2 domain (green) has no affinity to the PTP domain either, due in part to the bulkier residues of the bDbE loop (E176, L177), and also to the positively charged sidechain (K178) in the bEx48 position, which should be repulsed by the proximal lysine/arginine cluster.