Structure–function–folding relationship in a WW domain

Marcus Jäger

Yan Zhang

Jan Bieschke

Houbi Nguyen

*Department of Chemistry and The Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, BCC265, La Jolla, CA 92037;

^{Howard Hughes Medical Institute, The Jack H. Skirball Center for Chemical Biology and Proteomics, The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA 92037; and}

^{Center for Biophysics and Computational Biology and}

**Departments of Chemistry and Physics, University of Illinois, Urbana, IL 61801

Edited by Robert L. Baldwin, Stanford University Medical Center, Stanford, CA, and approved May 16, 2006

^{M.J. and Y.Z. contributed equally to this work.}

^{Present address: Department of Chemistry and Biochemistry, University of California, Los Angeles, CA 90024.}

^{Present address: Department of Chemistry, Stanford University, Stanford, CA 94305.}

^{To whom correspondence should be addressed. E-mail:}ude.sppircs@yllekj

Author contributions: M.J. and J.W.K. designed research; M.J., Y.Z., J.B., H.N., M.D., and M.E.B. performed research; J.P.N. and M.G. contributed new reagents/analytic tools; M.J., Y.Z., J.B., and H.N. analyzed data; and M.J., Y.Z., M.G., and J.W.K. wrote the paper.

Edited by Robert L. Baldwin, Stanford University Medical Center, Stanford, CA, and approved May 16, 2006

Received 2006 Jan 28

Abstract

Protein folding barriers result from a combination of factors including unavoidable energetic frustration from nonnative interactions, natural variation and selection of the amino acid sequence for function, and/or selection pressure against aggregation. The rate-limiting step for human Pin1 WW domain folding is the formation of the loop 1 substructure. The native conformation of this six-residue loop positions side chains that are important for mediating protein–protein interactions through the binding of Pro-rich sequences. Replacement of the wild-type loop 1 primary structure by shorter sequences with a high propensity to fold into a type-I′ β-turn conformation or the statistically preferred type-I G1 bulge conformation accelerates WW domain folding by almost an order of magnitude and increases thermodynamic stability. However, loop engineering to optimize folding energetics has a significant downside: it effectively eliminates WW domain function according to ligand-binding studies. The energetic contribution of loop 1 to ligand binding appears to have evolved at the expense of fast folding and additional protein stability. Thus, the two-state barrier exhibited by the wild-type human Pin1 WW domain principally results from functional requirements, rather than from physical constraints inherent to even the most efficient loop formation process.

Keywords: β-turn, ligand binding, protein folding, β-sheet, protein function

Abstract

Globular proteins evolve by mutation and selection. Selection criteria include function, and sufficient thermodynamic stability and folding rate to avoid sustained chaperone binding and proteasome degradation. The selection criteria cannot always be optimized independently over the entire sequence of a protein. For the human Pin1 (hPin1) WW domain (Pin WW hereafter), we have shown that residues important for stability and folding rate are segregated in the sequence (1 –4). It is likely that functional selection criteria are predominant once minimal energetic criteria are met. Therefore, sequence evolution to enhance function may lead to a decrease in protein stability and folding rate compared with a sequence optimized for folding energetics.

The hPin1 cell cycle regulatory proline (Pro) cis/trans-isomerase is a two-domain protein (5). In its physiological role, the N-terminal WW domain binds Pro-rich ligands of the consensus sequence (pS/pT)P, whereas the C-terminal domain catalyzes the Pro cis/trans-isomerization at the pS/pT-P peptide bond. NMR solution studies show that the two domains, which are connected by a flexible solvated linker, interact only weakly before ligand binding (6, 7). The structure of the isolated Pin WW domain is virtually superimposable on that of the WW domain in the two-domain hPin1 protein (8). Moreover, Pin WW exhibits sufficient thermodynamic stability for biophysical analysis, folds rapidly, and retains its ligand-binding function (3, 9). These attributes, combined with sequence information on >150 WW domain family members (10, 11), makes Pin WW an excellent small model protein for structure–function–folding studies.

WW domains have been used extensively in experimental and theoretical folding studies (1 –4, 12 –19). Traditional side-chain and amide-to-ester mutagenesis of Pin WW in conjunction with laser temperature-jump (T-jump) relaxation studies revealed that the folding rate was limited by nucleation at the loop 1 substructure (1 –3, 16), similar to the situation observed in SH3 domains (20, 21).

The six-residue loop 1 of Pin WW is unusually long. Sequence alignments of >150 WW domains reveal that a five-residue loop is statistically favored in this protein family, and structural studies on these loops indicate that a type-I G1 bulge conformation seems to be the preferred local structural motif (5, 22 –26). An x-ray structure of the full-length hPin1 cis/trans-isomerase revealed that loop 1 in the WW domain adopts an unusual conformation: a four-residue type-II turn intercalated within a larger, six-residue loop (5, 9) (Fig. 1a). NMR solution studies on the isolated Pin WW indicate that loop 1 is intrinsically flexible (8), whereas the topologically equivalent five-residue type-I G1 bulge turn in the FBP28 WW domain (FBP WW) (Fig. 1b) is structurally more ordered (22).

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Fig. 1.

Loop structures and sequences of WW domains. (a) Backbone diagram of the loop 1 substructure in WT Pin WW (residues S16–R21) [Protein Data Bank (PDB) ID code 1PIN]. (b) Backbone diagram of the loop 1 substructure in WT FBP WW (residues T13–K17) (PDB ID code 1E01). Backbone H-bonds are indicated by black dotted lines. (c) Aligned sequences of the WT Pin WW domain (variant 1) and loop 1 redesigned variants 2–9 and the redesigned and sequence-minimized FBP WW variants (10 and 11). β-strand residues are colored blue, residues that were mutated or deleted upon loop 1 redesign are in red, and all other residues are in gray.

Mutagenesis studies reveal that the side chain of Arg-17 in loop 1 of Pin WW contributes significantly to ligand-binding energy through recognition of a phosphorylated Ser residue (9). Replacing Arg-17 by Ala results in a 6.3-fold decrease in binding affinity toward the phosphorylated peptide YSPTpSPS, derived from the C-terminal domain of RNA polymerase II, a natural ligand of Pin WW (27). It is therefore possible that the sequence of the loop 1 substructure of Pin WW, with its unusual conformation and intrinsic dynamics, is not optimized for folding kinetics, but instead has been evolutionary optimized for functional duties.

To support this hypothesis, we must demonstrate that significantly faster folding and more stable variants of Pin WW can be made by shortening loop 1 and that such shortened loops result in lower ligand-binding affinity whether Arg-17 is present or not. Structural, thermodynamic, kinetic, and ligand-binding studies on loop 1 Pin WW variants affirm that functional attributes contribute substantially to the selection criteria for this region of Pin WW.

See Fig. 3 for more results. Var., variant no.

See Fig. 4 for more results. Var., variant no.

Click here to view.

Acknowledgments

M.G. and H.N. were supported by National Science Foundation Grant MCB 0316925. J.W.K., M.D., and M.J. were supported by National Institutes of Health Grant GM 051105, The Skaggs Institute of Chemical Biology, and the Lita Annenberg Hazen Foundation. M.J. was supported by postdoctoral fellowships from the Deutsche Forschungsgemeinschaft (DFG) and the La Jolla Interfaces in Science program. Y.Z. was supported by National Institutes of Health Grant CA 054418. J.P.N. is a Howard Hughes Medical Institute Investigator.

Acknowledgments

Abbreviations

hPin1	human Pin1
Pin WW	hPin1 WW domain
FBP WW	FBP28 WW domain
T-jump	temperature-jump.

Abbreviations

Footnotes

Conflict of interest statement: No conflicts declared.

This paper was submitted directly (Track II) to the PNAS office.

Data deposition: The atomic coordinates and structure factors have been deposited in the Protein Data Bank, www.pdb.org (PDB ID codes 1zcn and 2f21).

Footnotes

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