Influence of hPin1 WW N-terminal domain boundaries on function, protein stability, and folding

WW domains are naturally occurring protein–protein interaction modules of 30–50 residues, found in multidomain signaling and regulatory proteins (Sudol et al. 1995; Sudol 1996; Macias et al. 2000; Hu et al. 2004). Biophysical studies reveal that WW domains fold into their characteristic twisted three-stranded β-sheet architecture, and most (but not all) WW domains remain fully folded and functional when studied in isolation (Macias et al. 1996, 2000; Pires et al. 2001; Kowalski et al. 2002; Wiesner et al. 2002; Russ et al. 2005; Socolich et al. 2005; Petrovich et al. 2006). The human cell regulatory protein hPin1 is a 167 residue two-domain protein, composed of an N-terminal WW domain (residues 1–39, hPin1 WW hereafter) and a C-terminal cis/trans-isomerase domain (residues 45–167). The domains are connected by a flexible linker (Ranganathan et al. 1997). Isolated hPin1 WW 6–39 has been studied extensively by us experimentally (Jager et al. 2001, 2006; Deechongkit and Kelly 2002; Deechongkit et al. 2004, 2006; Nguyen et al. 2005; Powers et al. 2005) and others theoretically (Zhou 2003; Cheung et al. 2005; Cecconi et al. 2006). Its small size, high solubility (mM range) (Kowalski et al. 2002), aggregation resistance (M. Jager and J.W. Kelly, unpubl.), ready availability by chemical synthesis (Kaul et al. 2001) or recombinant expression, and its simple two-state unfolding (Jager et al. 2001, 2006) make it attractive as a model system to understand β-sheet folding energetics (Deechongkit et al. 2006). Moreover, it is amenable to extensive conventional side-chain mutagenesis as well as backbone amide mutagenesis (amide-to-ester or amide-to-E-olefin) enabling perturbations to be made to probe the basis for its stability and fast folding (microsecond timescale) (Jager et al. 2001, 2006; Kaul et al. 2001; Deechongkit and Kelly 2002; Deechongkit et al. 2004, 2006; Nguyen et al. 2005). Previous thermodynamic and kinetic studies on hPin1 WW were conducted with a sequence-minimized version (variant 5, Fig. 1B), which lacks the first five N-terminal residues (Met1-Ala-Asp-Glu-Glu5) (Jager et al. 2001, 2006). These residues were not structurally defined in the first high-resolution X-ray structure of full-length hPin1, and were assumed to be disordered (Ranganathan et al. 1997). However, in a more recent X-ray structure of hPin1 (1.3 A resolution), the negatively charged N-terminal Glu4 and Glu5 residues are structurally well defined and appear to be engaged in an ionic interaction with the positively charged ɛ-amino group of Lys13 in β-strand 1 (Fig. 1A; Verdecia et al. 2000). Lys13 is only weakly conserved among WW domain family members (more than 200 members). Position 13 residues with the highest frequencies (in parentheses) are Glu (0.32), Lys (0.15), Met (0.13), Arg (0.11), and Val (0.08). The N-terminal region is not conserved in the structurally and functionally related WW domains of the Pin1 cis/trans-isomerases from yeast or fungi (Fig. 1B, first three entries). To investigate the importance of ionic interactions between Glu4, Glu5, and Lys13 in the hPin1 WW ground and transition states, a detailed mutational study was conducted (Fig. 1B, variants 1–12). Figure 1. (A) Structural depiction of hPin1 WW (residues 1–39) generated using PDB-file 1F8A. The labeled side chains of residues Asp3, Glu4, Glu5, and Lys13 are shown in stick representation. (B) Sequence alignment of the hPin1 WW domain variants discussed ...