Increasing protein stability using a rational approach combining sequence homology and structural alignment: Stabilizing the WW domain.
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).
Jiang
X
X
Department of Chemistry and the Skaggs Institute of Chemical Biology, The Scripps Research Institute, 10550 Torrey Pines Road, La Jolla, CA 92037, USA.
Kowalski
J
J
Kelly
J W
JW
D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics; D018797:Cell Cycle Proteins; D015195:Drug Design; D004355:Drug Stability; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019696:Peptidylprolyl Isomerase / Q000737:chemistry / Q000235:genetics; D010750:Phosphoproteins / Q000737:chemistry* / Q000235:genetics; D011489:Protein Denaturation; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D017385:Sequence Homology; D013057:Spectrum Analysis; D014157:Transcription Factors; D000091102:YAP-Signaling Proteins
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D010750:Phosphoproteins; D014157:Transcription Factors; D000091102:YAP-Signaling Proteins; C088374:YAP1 protein, human; C486223:Yap1 protein, mouse; D019696:Peptidylprolyl Isomerase