Comparative Analysis of Mutant Tyrosine Kinase Chemical Rescue<xref ref-type="fn" rid="fn2"></xref><xref ref-type="fn" rid="fn1"></xref>

Protein tyrosine kinases are critical cell signaling enzymes. These enzymes have a highly conserved Arg residue in their catalytic loop which is present two residues or four residues downstream from an absolutely conserved Asp catalytic base. Prior studies on protein tyrosine kinases Csk and Src revealed the potential for chemical rescue of catalytically deficient mutant kinases (Arg to Ala mutations) by small diamino compounds, particularly imidazole; however, the potency and efficiency of rescue was greater for Src. This current study further examines the structural and kinetic basis of rescue for mutant Src as compared to mutant Abl tyrosine kinase. An X-ray crystal structure of R388A Src revealed the surprising finding that a histidine residue of the N-terminus of a symmetry-related kinase inserts into the active site of the adjacent Src and mimics the hydrogen-bonding pattern seen in wild-type protein tyrosine kinases. Abl R367A shows potent and efficient rescue more comparable to Src, even though its catalytic loop is more like that of Csk. Various enzyme redesigns of the active sites indicate that the degree and specificity of rescue are somewhat flexible, but the overall properties of the enzymes and rescue agents play an overarching role. The newly discovered rescue agent 2-aminoimidazole is about as efficient as imidazole in rescuing R/A Src and Abl. Rate vs pH studies with these imidazole analogues suggest that the protonated imidazolium is the preferred form for chemical rescue, consistent with structural models. The efficient rescue seen with mutant Abl points to the potential of this approach to be used effectively to analyze Abl phosphorylation pathways in cells. Protein tyrosine kinases (PTKs) are key enzymes in cell signaling and play roles in a wide range of diseases including cancer (1, 2). Several PTKs, including Abl, are targeted clinically with therapeutic agents, and others are the subject of pharmaceutical development (3). As members of the protein kinase superfamily, PTKs share conserved sequences andfolds(2),andyet theyshowdistinctsubstrateselectivity(4,5) and modes of regulation (6). Evidence suggests that the mechanism of PTK-catalyzed phosphoryl transfer is dissociative, in which the bond to the leaving group ADP is largely broken prior to tyrosine phenol attack on the γ-phosphorus of ATP (7). The alignment, orientation, and spacing of the tyrosine and ATP substrates by key active site residues are believed to be crucial in facilitating catalysis based on structural (8), mutagenic (9), and kinetic (10) studies. The catalytic loop sequence, DLAARN, is conserved throughout the vast majority of the 90 human PTKs but differs in the nine members of the Src family, which have the sequence DLRAAN (Figure 1A) (2). This relocation of the Arg from the D+4 position in most PTKs to the D+2 position in the Src family suggests an unusual functional plasticity. A crystal structure of an insulin receptor tyrosine kinase-bisubstrate analogue complex is believed to capture the active conformation of the enzyme and reveals the orientation of the active site residues (8). This structure shows that the side chain of the catalytic loop Asp makes a hydrogen bond to the tyrosine hydroxyl and likely serves as the catalytic base. The catalytic loop Arg side chain makes apparent hydrogen bonds to this Asp side chain, as well as the substrate phenol oxygen. This triangle of hydrogen bonds (Figure 1B) appears to provide a scaffold that aligns the reactants. It is assumed that the Src Arg residue, located in the noncanonical D+2 position, fulfills a similar role to that of the catalytic loop Arg in the D+4 position in non-Src PTKs. Mutagenic analysis of the catalytic loop Arg has been performed previously on PTK Csk (11) and Src (12). Mutation of the D+4 Arg in PTK Csk to Ala (R318A Csk) resulted in a dramatic kcat drop of 3000-fold with relatively † This work was supported by National Institutes of Health Grant CA74305 (P.A.C.), the Johnson & Johnson Fellowship of Life Science Research (M.A.S.), and the Jane Coffin Childs Memorial Fund for Medical Research (J.J.H.). ‡ The PDB code for the structure here is 3GEQ. * To whom correspondence should be addressed. Tel: 410-614-8849. Fax: 410-614-7717. E-mail: [email protected]. § These authors contributed equally to this work. | Johns Hopkins University School of Medicine. ⊥ Current address: Department of Chemistry, American University, Washington, DC 20016. # University of California, Berkeley. O University of Washington. ∇ Howard Hughes Medical Institute. 1 Abbreviations: AblKD, human c-Abl kinase domain; D+2, PTK with active loop sequence DLRAAN; D+4, PTK with active loop sequence DLAARN; PP2, 4-amino-5-(4-chlorophenyl)-7-(tert-butyl)pyrazolo[3,4-d]pyrimidine; PTK, protein tyrosine kinase; SrcKD, chicken c-Src kinase domain. Biochemistry 2009, 48, 3378–3386 3378 10.1021/bi900057g CCC: $40.75  2009 American Chemical Society Published on Web 03/04/2009 small (<10-fold) Km changes. A Csk double mutant was prepared to mimic the Src-like catalytic loop (D+2 Arg Csk), and this mutant showed a 150-fold increased kinase activity compared with R318A Csk (11). While still 20-fold less than wild type, the substantial kinase activity of the D+2 Arg Csk mutant bolsters the argument for functional plasticity of the catalytic loop Arg residue. In some cases, enzyme active site point mutants can be chemically rescued by small molecules that complement the deleted side chains (13-15). Indeed, chemical rescue of R318A Csk with a series of diamino compounds (guanidiniums, amidiniums, imidazoles) could restore substantial activity to this mutant (11). Imidazole was the most efficient R318A Csk rescue agent, showed Km ) 20 mM, and stimulated kinase activity up to the level of the D+2 Arg Csk. Related studies were carried out on Src, and it was shown that mutation of the D+2 Arg in Src to Ala (R388A Src) is 200-fold less active than wild type (12). A Src double mutant that mimics the canonical catalytic loop (D+4 Arg Src) was 2-fold more active than wild type. Imidazole rescued R388A Src to a level of 50% of wild type, with a Km ) 5 mM. The ability to rescue R318A Csk and R388A Src to 5-50% of wild-type kcat values with a nontoxic concentration of imidazole (<50 mM) raised the interesting possibility that it might be feasible to complement these mutant tyrosine kinases in cells. Indeed, it was demonstrated that imidazole could restore Csk and Src functionality to mouse embryonic fibroblasts expressing replacement Arg f Ala mutant kinases (12, 16). Using this chemical rescue approach, new insights into the rapid signaling events have been obtained for Src and Csk (12, 17, 18). Although progress on the chemical rescue of mutant Src and Csk has been made, questions remain. Does the imidazole occupy an active site position mimicking the position of the catalytic loop Arg side chain? Are Arg f Ala mutant PTKs beyond Src and Csk likely to show more efficient chemical rescue, like Src, or less efficient rescue, like Csk? Can alternative potent rescue combinations of kinase mutations and small molecules be identified? Is the imidazole interacting with the mutant kinase as the imidazolium or the neutral species? Here we use a combination of X-ray crystallography and extended mutagenic and kinetic experiments on Abl and Src to address these questions. EXPERIMENTAL PROCEDURES Rosetta Design Calculations. The crystal structure of the insulin receptor tyrosine kinase (PDB code 1IR3) (19) was used as the scaffold for design calculations. Because the binding site for the small molecules used for chemical rescue was unknown, atoms comprising the guanidinium group of the mutated arginine residue (corresponding to R388 in chicken c-Src) were converted into a fixed ligand. Alternate amino acids were selected at positions corresponding to W428 and E454 in chicken c-Src. Side-chain conformational freedom was modeled with a backbone-dependent rotamer library (20) supplemented by extra sample points with 1 and 2 values ( 1 standard deviation in the observed torsional distributions. A Monte Carlo optimization algorithm was used to identify sequences and conformations that minimize the Rosetta full-atom energy potential of the complex (21). Cloning, Expression, and Purification. Src and Abl variants were generated following QuikChange protocols, with the addition of 2% DMSO to the reaction mixture. The targeted mutagenesis was confirmed by DNA sequencing. Src variants used in enzymatic assays were expressed as previously described (12). These preparations consisted of residues 85-533 of chicken c-Src. Double and triple mutants of R388A Src plus mutations at position 454 to alanine, lysine, or arginine and/or at position 428 to glutamate or glutamine resulted in very low expression levels and were not purified. Double mutants of R388A Src plus mutations at 428 to tyrosine, phenylalanine, and alanine were purified as previously described (12). For the purposes of crystallization, the R388A mutant of the kinase domain of chicken c-Src (R388A SrcKD; residues 251-533) (22) was expressed and purified in Escherichia coli (23). R388A SrcKD was purified fused to an N-terminal TEV-cleavable His6 tag, which results in a Gly-His-Met sequence before the start of the kinase domain sequence. The identity of the purified protein was confirmed by mass spectrometry (David King, Howard Hughes Medical Institute, Berkeley) and was found to be of the calculated mass without any posttranslational modification. The previously described protocol for expression and purification of the Abl kinase domain (AblKD; residues 229-511 of human c-Abl) (23) was modified for the variants described here. By coexpressing AblKD with the catalytic domain of YopH phosphatase, instead of the full-length phosphatase, copurification of YopH and AblKD was mitigated. Therefore, only a single purification step with a Ni affinity column was performed. DTT (2 mM) was added to all purification steps, and the histidine tag was not cleaved prior to kinetic measurements. The catalytic domain of YopH phosphatase (corresponding to residues 164-468) was subcloned with Pfu Turbo FIGURE 1: Catalytic loop of selected kinases. (A) Amino acid sequence of the catalytic loop region for Src (residues 384-391), Abl (residues 361-368), Csk, insulin receptor tyrosine kinase (IRK), and epidermal growth factor receptor kinase (EGFR). The D+2 Arg and D+4 Arg that are examined here are in bold. (B) Schematic of the interaction of the catalytic loop residues corresponding to Asp363 and Arg367 in Abl with the substrate tyrosine, as observed in the crystal structure of inhibited IRK. Comparison of Tyrosine Kinase Chemical Rescue Biochemistry, Vol. 48, No. 15, 2009 3379