br Target enzyme attributes and substrate peptide selection
Target enzyme attributes and substrate peptide selection Protein kinases catalyze the phosphorylation of serine, threonine, and tyrosine residues in both proteins and peptides using ATP as the phosphoryl donor. The human kinome is comprised of 518 protein kinases and 40 lipid kinases. The vast majority (478) of the former contain the so-called eukaryotic protein kinase (EPK) domain, a stretch of approximately 250 residues that encompass the catalytic region (Duong-Ly & Peterson, 2013; Kostich et al., 2002). The substrate specificity of the large EPK family is controlled by three key determinants: (1) The active site substrate specificity defines the ability of a protein kinase to phosphorylate one or more alcohol-bearing residues in the active site. Indeed, the EPKs are divided into three distinct groups based on their active site specificity: the tyrosine proteins kinases (TPKs), the serine/threonine protein kinases (SPKs), and the so-called dual specificity protein kinases that catalyze the phosphorylation all three types of alcohol-bearing residues (Miller & Turk, 2018). However, the active site specificity of protein kinases is not limited to the three genetically encoded alcohol-containing amino acids. A wide variety of unnatural residues are phosphorylated by protein kinases, including a structurally constrained tyrosine residue [(7-(S)-hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylic FSLLRY-NH2 (Htc)] (Kwon, Mendelow, & Lawrence, 1994; Kwon et al., 1993; Lee, Niu, & Lawrence, 1995; Prorok, Sukumaran, & Lawrence, 1989; Turner et al., 2016). Substrates containing Htc are particularly useful as probes of TPK activity since the corresponding phosphorylated product is resistant to dephosphorylation by intracellular protein phosphatases (Turner et al., 2016). One of the key advantages of using peptides as protein kinase substrates is that unnatural residues are readily introduced during peptide synthesis. Non-naturally occurring residues have been used to endow peptide-based substrates with useful properties, including enhanced selectivity for specific protein kinases, resistance to intracellular proteolysis (vide infra), and photo-transformation from inactive to active substrates. (2) The sequence substrate specificity of a protein kinase defines its preference for the amino acid sequence encompassing the phosphorylatable residue. This is also referred to as the consensus sequence of a protein kinase. This form of substrate specificity was initially derived from sequencing studies on intact phosphorylated protein substrates. However, starting in the 1990s, peptide-based libraries have been used to define the sequence specificity of individual protein kinases. Although these studies have generally been limited to the use of the standard genetically encoded amino acids, the sequence specificity of protein kinases extends well beyond the conventional 20 amino acids. For example, we have used a combination of D- and N-(Me) amino acids at various sites along the peptide chain to create effective protein kinase substrates that are resistant to proteolysis (Mainz, Serafin, et al., 2016; Mainz, Wang, Lawrence, & Allbritton, 2016; Proctor et al., 2012a, Proctor et al., 2012b; Proctor, Zigoneanu, Wang, Sims, & Lawrence, 2016). (3) Multisite substrate specificity describes interactions between a protein kinase and its substrate that occur outside of the kinase catalytic cleft. These ancillary protein-protein interactions are used to dock the protein substrate near the appropriate protein kinase. For example, the substrate specificity of different members of the mitogen-activated protein kinases is determined by their ability to recognize so-called D-site or DEF-site motifs on their substrates, an interaction that occurs distal from the active site region (Miller & Turk, 2018). Ancillary non-active site interactions can be recapitulated in peptides by appending the secondary recognition motif to the consensus sequence via a linker region (Profit, Lee, Niu, & Lawrence, 2001).