Characterization of the SRP68/72 interface of human signal recognition particle by systematic site-directed mutagenesis.
The signal recognition particle (SRP) is a ribonucleoprotein complex which is crucial for the delivery of proteins to cellular membranes. Among the six proteins of the eukaryotic SRP, the two largest, SRP68 and SRP72, form a stable SRP68/72 heterodimer of unknown structure which is required for SRP function. Fragments 68e' (residues 530 to 620) and 72b' (residues 1 to 166) participate in the SRP68/72 interface. Both polypeptides were expressed in Escherichia coli and assembled into a complex which was stable at high ionic strength. Disruption of 68e'/72b' and SRP68/72 was achieved by denaturation using moderate concentrations of urea. The four predicted tetratricopeptide repeats (TPR1 to TPR4) of 72b' were required for stable binding of 68e'. Site-directed mutagenesis suggested that they provide the structural framework for the binding of SRP68. Deleting the region between TPR3 and TPR4 (h120) also prevented the formation of a heterodimer, but this predicted alpha-helical region appeared to engage several of its amino acid residues directly at the interface with 68e'. A 39-residue polypeptide (68h, residues 570-605), rich in prolines and containing an invariant aspartic residue at position 585, was found to be active. Mutagenesis scanning of the central region of 68h demonstrated that D585 was solely responsible for the formation of the heterodimer. Coexpression experiments suggested that 72b' protects 68h from proteolytic digestion consistent with the assertion that 68h is accommodated inside a groove formed by the superhelically arranged four TPRs of the N-terminal region of SRP72.
Iakhiaeva
Elena
E
Department of Molecular Biology, University of Texas Health Science Center at Tyler, 75708, USA.
Hinck
Cynthia S
CS
Hinck
Andrew P
AP
Zwieb
Christian
C
D001665:Binding Sites / Q000235:genetics / Q000502:physiology; D006801:Humans; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D011485:Protein Binding / Q000502:physiology; D018271:Signal Recognition Particle / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014508:Urea / Q000737:chemistry
D050505:Mutant Proteins; C512269:SRP68 protein, human; C503636:SRP72 protein, human; D018271:Signal Recognition Particle; D014508:Urea
Nature. 2004 Feb 26;427(6977):808-14
14985753
Bioinformatics. 2000 Apr;16(4):404-5
10869041
EMBO J. 1987 Nov;6(11):3471-7
2828031
J Mol Biol. 1994 Mar 11;236(5):1407-9
8126729
Bioinformatics. 2004 Feb 12;20(3):426-7
14960472
J Cell Sci. 2005 Oct 1;118(Pt 19):4551-62
16179612
Proc Natl Acad Sci U S A. 2003 Apr 29;100(9):5142-7
12702764
Curr Opin Struct Biol. 2005 Feb;15(1):116-25
15718142
Nucleic Acids Res. 2008 Jul 1;36(Web Server issue):W197-201
18463136
Cell. 1983 Sep;34(2):525-33
6413076
Nucleic Acids Res. 2006 Jan 1;34(Database issue):D163-8
16381838
J Cell Biol. 1981 Nov;91(2 Pt 1):545-50
7309795
Sci STKE. 2003 Apr 22;2003(179):RE8
12709533
FEBS Lett. 2005 Feb 7;579(4):921-6
15680975
Biochim Biophys Acta. 2004 Nov 11;1694(1-3):17-35
15546655
RNA Biol. 2008 Apr-Jun;5(2):73-83
18418087
Eur J Biochem. 1987 Mar 16;163(3):519-28
3030745
EMBO J. 1994 Sep 15;13(18):4390-400
7925282
Bioessays. 1999 Nov;21(11):932-9
10517866
EMBO J. 1998 Mar 2;17(5):1192-9
9482716
Protein Sci. 2006 Jun;15(6):1290-302
16672232
Exp Cell Res. 2007 Feb 15;313(4):834-47
17239854
J Mol Biol. 2007 Mar 16;367(1):187-203
17254600
RNA Biol. 2007 Nov;4(3):154-9
18347438
Archaea. 2002 Mar;1(1):27-34
15803656
RNA. 2005 Jan;11(1):7-13
15611297
Nature. 2000 Nov 9;408(6809):167-73
11089964
Nucleic Acids Res. 2004 Jul 1;32(Web Server issue):W20-5
15215342
Proc Natl Acad Sci U S A. 2000 Jan 4;97(1):55-60
10618370
J Mol Biol. 2005 Jan 28;345(4):659-66
15588816
Cell. 2000 Apr 14;101(2):199-210
10786835
RNA. 2008 Jun;14(6):1143-53
18441046
Annu Rev Biochem. 2004;73:539-57
15189152
Nucleic Acids Res. 2004 Mar 19;32(5):1792-7
15034147