Increasing protein stability using a rational approach combining sequence homology and structural alignment: Stabilizing the WW domain

Abstract

This study shows that a combination of sequence homology and structural information can be used to increase the stability of the WW domain by 2.5 kcal mol(-1) and increase the T(m) by 28 degrees C. Previous homology-based protein design efforts typically investigate positions with low sequence identity, whereas this study focuses on semi-conserved core residues and proximal residues, exploring their role(s) in mediating stabilizing interactions on the basis of structural considerations. The A20R and L30Y mutations allow increased hydrophobic interactions because of complimentary surfaces and an electrostatic interaction with a third residue adjacent to the ligand-binding hydrophobic cluster, increasing stability significantly beyond what additivity would predict for the single mutations. The D34T mutation situated in a pi-turn possibly disengages Asn31, allowing it to make up to three hydrogen bonds with the backbone in strand 1 and loop 2. The synergistic mutations A20R/L30Y in combination with the remotely located mutation D34T add together to create a hYap WW domain that is significantly more stable than any of the protein structures on which the design was based (Pin and FBP28 WW domains).

Fig. 1.

Sequence of the hYap, Pin, and FBP28 WW domains (top) as well as the consensus sequence generated by the SMART server (http:// smart.embl-heidelberg.de) (bottom). Wave-underlined residues compose hydrophobic cluster 1 in the hYap sequence, whereas doubly underlined residues belong to hydrophobic cluster 2 (see text). Boldface letters identify the residues at positions 20, 30, and 34 in the hYap WW domain, which are mutated to increase the protein stability. In the consensus sequences, the single-letter amino acid codes are in capital letters; lowercase letters are defined as follows: (h) hydrophobic; (a) aromatic; (p) polar; (o) alcohol (Ser or Thr); (t) turn-like residues (Ala, Cys, Asp, Glu, Gly, His, Lys, Asn, Gln, Arg, Ser, and Thr). Positions with low or no sequence conservation are marked with a dot. The definitions were slightly modified from the original output of SMART program to improve readability. Residues involved in either of the hydrophobic clusters that show ≥50% consensus but are not highly conserved are defined as semi-conserved. This study focuses on these semi-conserved core residues and their surrounding residues.

Fig. 2.

Structural comparison of the environment around the identically located Asn31 in hYap (A) and Asn26 in Pin (B). The numbers on dotted lines indicate non-bonded distances in Å between the two heavy atoms. These asparagine residues adopted different χ₂ angles, resulting in different hydrogen bonding patterns around them (see text for more detail).

Fig. 3.

Far-UV CD spectra of the wild-type hYap WW domain (50 μM) at 2°C (·) and at 98°C (+), as well as that of the A20R/L30Y/D34T (▪) and N31A (Δ) WW domains at 2°C. The N31A variant is mostly unfolded, implying the importance of Asn31 for structural integrity at pH 7.

Fig. 4.

Structural comparison of the ligand-binding hydrophobic cluster 2 in hYap (A) and Pin WW (B) domains. The residues composing cluster 2 are depicted by a ball-and-stick representation, with backbone atoms in white, whereas the side-chain atoms are represented with darker shades. The figures were generated using Molscript (Kraulis 1991), on the basis of the NMR structure of hYap (Macias et al. 1996) and the crystal structure of the WW domain from the Pin (Ranganathan et al. 1997, PDB file 1pin).

Fig. 5.

NMR (1D proton) of the amide region of the wild type (A), A20R (B), L30Y (C), D34T (D), and A20R/L30Y/D34T (E). Notice the well-dispersed chemical shifts for the variants. The two dotted lines indicate chemical shifts of the two labeled tryptophan indole NHs in the wild-type hYap WW domain (10.48 and 10.16 ppm).

Fig. 6.

Thermal denaturation curves of hYap wild-type and variant WW domains. (A) Molar ellipticity at 230 nm and (B) percentage of unfolding as a function of temperature. The identities of the curves are as follows: (·) wild type; (|ap) L30Y; (×) D34T; (○) A20R; (♦) A20R/D34T; (▴) A20R/L30Y; and (▪) A20R/L30Y/D34T. Percentage of unfolding was calculated according to equation 4 after fitting the original data to equations 2 and 3. Solid lines through the data are the fitting results for (A) and smoothed curves to facilitate viewing for (B).

Fig. 7.

GdnHCl denaturation of hYap wild-type and variant WW domains. (A) Near-UV CD scans of A20R/L30Y/D34T in pH 7 buffer (4°C) (·), 6 M GdnHCl (○), and at 100°C (×). (B) Fluorescence emission scans of A20R/L30Y/D34T in buffer (solid line) and 6 M GdnHCl (dashed line). (C) GdnHCl denaturation curves determined by Trp fluorescence emission at 345 nm (4°C) with samples excited at 295 nm. The identities of the curves are as follows: (·) wild-type; (|ap) L30Y; (×) D34T; (○) A20R; (♦) A20R/D34T; (▴) A20R/L30Y; and (▪) A20R/L30Y/D34T. Solid lines are fitted to equation 1.

Fig. 7.

GdnHCl denaturation of hYap wild-type and variant WW domains. (A) Near-UV CD scans of A20R/L30Y/D34T in pH 7 buffer (4°C) (·), 6 M GdnHCl (○), and at 100°C (×). (B) Fluorescence emission scans of A20R/L30Y/D34T in buffer (solid line) and 6 M GdnHCl (dashed line). (C) GdnHCl denaturation curves determined by Trp fluorescence emission at 345 nm (4°C) with samples excited at 295 nm. The identities of the curves are as follows: (·) wild-type; (|ap) L30Y; (×) D34T; (○) A20R; (♦) A20R/D34T; (▴) A20R/L30Y; and (▪) A20R/L30Y/D34T. Solid lines are fitted to equation 1.

Fig. 7.

GdnHCl denaturation of hYap wild-type and variant WW domains. (A) Near-UV CD scans of A20R/L30Y/D34T in pH 7 buffer (4°C) (·), 6 M GdnHCl (○), and at 100°C (×). (B) Fluorescence emission scans of A20R/L30Y/D34T in buffer (solid line) and 6 M GdnHCl (dashed line). (C) GdnHCl denaturation curves determined by Trp fluorescence emission at 345 nm (4°C) with samples excited at 295 nm. The identities of the curves are as follows: (·) wild-type; (|ap) L30Y; (×) D34T; (○) A20R; (♦) A20R/D34T; (▴) A20R/L30Y; and (▪) A20R/L30Y/D34T. Solid lines are fitted to equation 1.

Fig. 8.

Peptide ligand (EYPPYPPPPYPSG) binding to the hYap wild-type and variant WW domains was monitored by fluorescence emission intensity at 345 nm. The ligand (1 mM) was titrated into wild-type (·), L30Y (|ap), D34T (×), A20R (○), and A20R/D34T (♦) WW domains at a concentration of 9 μM. Solid lines are fits to equation 6.