Contributions of conserved TPLH tetrapeptides to the conformational stability of ankyrin repeat proteins.
Ankyrin repeat (AR) proteins are one of the most abundant classes of repeat proteins and are involved in numerous physiological processes. These proteins are composed of various numbers of AR motifs stacked in a nearly linear fashion to adopt an elongated and nonglobular architecture. One salient feature prevalent in such a structural unit is the TPLH tetrapeptide or a close variant, T/SxxH, which initiates the helix-turn-helix conformation and presumably contributes to conformational stability through a hydrogen-bonding network. In the present study, we investigated the roles of T/SxxH motif in the stability, structure, and function of AR proteins by a systematic and rationalized mutagenic study on, followed by biochemical and biophysical characterization of, gankyrin, an oncogenic protein composed of seven ARs and six T/SxxH tetrapeptides, and P16, a tumor suppressor with four ARs but no TPLH tetrapeptide. Our results showed that this tetrapeptide is ineffectual on global structure and function, but contributes significantly to conformational stability when its stabilizing potentials are fully realized in the local conformation, including (1) the intra-AR hydrogen bonding involving the hydroxyl group; (2) the intra-AR and inter-AR hydrogen bonds involving the imidazole ring; and (3) the hydrophobic interaction associated with the Thr-methyl group. Considering that the capping and close-to-capping units tend to have more sequence diversity and more conformational variation, it could be also generally true that a T/SxxH motif close to the terminal repeats contributes little or even negatively to stability with respect to Ala substitution, but substantially stabilizes the global conformation when located in the middle of a long stretch of ARs.
Guo
Yi
Y
Ohio State Biochemistry Program, The Ohio State University, Columbus, OH 43210, USA.
Yuan
Chunhua
C
Tian
Feng
F
Huang
Kun
K
Weghorst
Christopher M
CM
Tsai
Ming-Daw
MD
Li
Junan
J
D000595:Amino Acid Sequence; D017089:Ankyrin Repeat; D051358:Cyclin-Dependent Kinase 4 / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D006860:Hydrogen Bonding; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000737:chemistry* / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex / Q000737:chemistry / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000737:chemistry / Q000378:metabolism
D009842:Oligopeptides; C403591:PSMD10 protein, human; D011518:Proto-Oncogene Proteins; D051358:Cyclin-Dependent Kinase 4; D046988:Proteasome Endopeptidase Complex
Trends Biochem Sci. 1999 Aug;24(8):311-6
10431175
Protein Sci. 2004 Jan;13(1):32-9
14691219
Science. 1988 Jun 17;240(4859):1648-52
3381086
J Mol Biol. 2007 Nov 2;373(4):990-1005
17881001
Structure. 2005 Aug;13(8):1131-41
16084385
PMC Biophys. 2009 Jun 26;2(1):5
19558684
Proteins. 2004 Apr 1;55(1):214-7
14997555
Trends Cell Biol. 2006 May;16(5):229-33
16581249
J Mol Biol. 2007 Jan 26;365(4):1201-16
17174335
Biochemistry. 2004 Sep 28;43(38):12152-61
15379554
Biochim Biophys Acta. 2002 Mar 14;1602(1):73-87
11960696
Proc Natl Acad Sci U S A. 2003 Feb 18;100(4):1700-5
12566564
Methods Enzymol. 1986;131:266-80
3773761
Biochemistry. 2009 May 19;48(19):4050-62
19320462
Cancer Cell. 2005 Jul;8(1):3-4
16023592
Protein Sci. 1995 Jul;4(7):1325-36
7670375
J Am Chem Soc. 2008 Apr 30;130(17):5681-8
18396879
Mol Cell. 1998 Feb;1(3):421-31
9660926
J Mol Biol. 2003 Mar 14;327(1):285-97
12614625
J Mol Biol. 1999 Nov 19;294(1):201-11
10556039
Biochemistry. 1999 Mar 9;38(10):2930-40
10074345
Biochemistry. 1996 Jul 23;35(29):9475-87
8755727
J Mol Biol. 2003 Sep 12;332(2):489-503
12948497
J Mol Biol. 2008 Feb 8;376(1):241-57
18164721
J Mol Biol. 2007 Jan 26;365(4):1187-200
17067634
Protein Sci. 2004 Nov;13(11):2864-70
15498935
Cancer Cell. 2005 Jul;8(1):75-87
16023600
Biochemistry. 1997 Jun 3;36(22):6633-42
9184143
Biochemistry. 2002 Mar 26;41(12):3977-83
11900540
Chembiochem. 2004 Feb 6;5(2):183-9
14760739
Nat Biotechnol. 2004 May;22(5):575-82
15097997
Protein Sci. 2004 Jun;13(6):1435-48
15152081
Biochemistry. 2003 Nov 25;42(46):13476-83
14621993
J Biol Chem. 2005 Jul 1;280(26):24715-22
15851475
Annu Rev Biochem. 1993;62:139-60
8352587
Biochemistry. 2006 Dec 26;45(51):15168-78
17176038