Cell, Vol. 64, 483-484, February8, 1991, Copyright© 1991 by Cell Press

Letter to the Editor

A Novel Cysteine-Rich Sequence Motif A number of protein sequence/structure motifs that bind to DNA have been elucidated from a series of X-ray crystallographic, nuclear magnetic resonance, and molecular biological analyses of protein-DNA complexes (for recent reviews see Latchman, 1990; Steitz, 1990). These include the classical zinc finger motif, the helix-turn-helix structure, and the helix-loop-helix motif, all of which have been shown to interact specifically with DNA. However, there is an increasing number of protein sequences for DNAbinding proteins where no obvious sequence and/or structure motif is apparent (for example, the SRF family of transcription factors). This would therefore suggest that there are a number of structures and/or sequences that have a role in DNA binding and recognition but as yet remain undiscovered. We would like to draw attention to a novel cysteine-rich amino acid sequence motif common to a number of diverse proteins thought to interact with DNA. The gene products from the human V(D)J recombination-activating gene RAG-1 (Schatz et al., 1989), RAD18 gene from Saccharomyces cerevisiae (Jones et al., 1988), IEl10 gene from herpes simplex virus (Perry et al., 1986), gene 61 from varicella-zoster virus (Davison and Scott, 1986), CG30 gene from baculovirus (Thiem and Miller, 1989), human ret transforming gene (Takahashi et al., 1988), and human rpt-1 interleukin 2 receptor regulator gene (Patarca et al., 1988) have not previously been considered to be related in amino acid sequence, but all contain the reported motif (see figure). The motif can be represented as C-X-(I,V)-C-X(11-30)-CX-H-X-(F,I,L)-C-X(2)-C-(I,L,M)-X(10-18)-C-P-X-C and is found only in seven I~roteins represented in the OWL data base, which indicates that the motif is highly specific. By chance alone, the calculated probability of finding the pattern in the whole OWL data base is 1.5 x 10-5, which is statistically highly significant.

The pattern resembles the well-known zinc finger motifs (Berg, 1990), although distinct differences are observed in the spacing between cysteine-cysteine/histidine pairs (usually 6 to 13 residues in zinc finger proteins) and also in the linking region between the two "finger-like" domains, which is unusually short (CXHXXCXXC). Any potential zinc/divalent metal-binding ligands from the central linking region would therefore be in close proximity to each other, and the motif could be interpreted as a modified CCCH-CCCC (nomenclature from Berg, 1990) or CCHC-CCCC two-finger sequence. This suggests a novel three-dimensional structure for the motif, as it is difficult to see how the potential divalent metal-binding ligands and the variable intervening linking regions could fit into known zinc finger structural frameworks. The proteins that are related by this novel motif are suggested to interact with DNA and are involved in diverse functions including site-specific recombination, DNA repair, and transcriptional regulation. No direct evidence for the involvement of the cysteine-rich motif in DNA binding has as yet been presented. However, we suggest that this conserved pattern of amino acids may represent a novel structure that binds zinc/divalent metal ions and DNA, analogous to other well-characterized zinc finger-containing proteins, including TFIIIA (Klug and Rhodes, 1987) and the glucocorticoid receptor family (Hard et al., 1990). If this is the case, then specificity of DNA interaction could be achieved by alteration of the length and sequence of the linking regions between the cysteine-cysteine/histidine pairs. The arrangement of the conserved cysteines, hydrophobic residues, proline, and histidine could then provide a structural framework for binding of zinc/divalent metal ions and DNA interaction in a variety of different DNA-interacting proteins. Another possibility is that the motif binds zinc/divalent metal ions to form a structure that is involved in specific protein-protein interaction, as proposed for the zinc-cysteine clusters of the adenovirus E1A and bacteriophage T4 gene 32 proteins (LUlie et al., 1987; Chatterjee et al., 1988; Pan et al., 1989). It will be of great

RAG-I

KSISCQICE

HILADPV

ETNCKHVFCRVCILRCLKVMGS

YCPSCRYPCFP

RADI8

TLLRCHICK

DFLKVPV

LT P C G H T F C S L C I R T H L N N Q P N

CPLCLFEFRE

IEII0

EGDVCAVCT

DEIAPHLRC

DTFPCMHRFCIPCMK TWMQLRN

TCPLCNAKLVY

VZ61

SDNTCTICM

STVSDLG

KTMPCLHDFCFVCIR AWTSTSV

QCPLCRCPVQ$

CG30

VKLQCNICFSVAEIKNYFLQPIDRLTIIPVLELDTCKHQLCSMCIRKIRKRKKV

PCPLCRVESLH

RET

QETTCPVCL

QYFAEPM

ML D C G H N I C C A C L A R C W G T A E T

RPT-I

EEVTCPICL

ELLKEPV

SA D C N H S F C R A C I T L N Y E S N R N T D G K G N C P V C R V P Y P F

RING1

SELMCPICL

DMLKNTM

TTKECLHRFCSDCIVTALRSGNK

NVSCPQCRETFPQ

ECPTCRKKLVS

Sequence Alignmentof ProteinsThat Containthe Cysteine-RichMotif The residues in bold type constitutethe novel cystaine motif (see text for details and references).RING1 is a novel gene about 95 kb centmmeric of the DP region of the human major histocompatibilitycomplex (Hanson et el., submitted). The motif was elucidatedfrom initial seamhas of the OWL (9.0) protein sequencedata bank of 25,049entries containing7,308,377residueson an AMT-distributedarray processorcomputer (Collinset al., 1988)using the amino acid sequenceof RING1.The motif was further refined using a pattern-matchingprogram written by Dr. M. Sternberg.

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interest to ascertain a functional role for this n e w motif. However, the statistically significant c o n s e r v a t i o n of the d e s c r i b e d s e q u e n c e pattern, from a v a r i e t y of proteins of diverse function a n d source, s u g g e s t s an i m p o r t a n t biological role. Paul S. F r e e m o n t , * I s a b e l M, H a n s o n , t and J o h n T r o w s d a l e t * Protein Structure L a b o r a t o r y tHuman Immunogenetics Laboratory Imperial C a n c e r R e s e a r c h F u n d L o n d o n W C 2 A 3PX England References Berg, J. M. (1990). J. Biol. Chem. 265, 6513-6516. Collins, J. F., Coulson, A. F. W., and Lyall, A. (1988). CABIOS 4, 67-71. Chatterjee, P. K., Bruner, M., Flint, S. J., and Harter, M. L. (1988). EMBO J. 7, 835-841. Davison, A. J., and Scott, J. E. (1986). J. Gen. Virol. 67, 1759-1816. Hard, T., Kellenbach, E., Boelens, R., Maler, B. A., Dahlman, K., Freedman, L. P., Carlstedt-Duke, J., Yamamoto, K. R., Gustafsson, J.-A., and Kaptein, R. (1990). Science 249, 157-160. Jones, J. S., Weber, S., and Prakash, L. (1988). Nucl. Acids Res. 15, 7119-7131. Klug, A., and Rhodes, D. (1987). Trends Biochem. Sci. 12, 464-469. Latchman, D. S. (1990). Biochem. J. 270, 281-289. Lillie, J. W., Loewenstein, P. M., Green, M. R., and Green, M. (1987). Cell 50, 1091-1100. Pan, T., Giedroc, D. R, and Coleman, J. E. (1989). Biochemistry 28, 8828-8832. Patarca, R., Schwartz, J., Singh, R. P., Kong, Q.oT., Murphy, E., Anderson, Y., Sheng, F.-Y.W., Singh, P., Johnson, K. A., Guarnagia, S. M., Durfee, T., Blattner, F., and Cantor, H. (1988). Proc. Natl. Acad. Sci. USA 85, 2733-2737. Perry, L. J., Rixon, F. J., Everett, R. D., Frame, M. C., and McGeoch, D. J. (1986). J. Gen. Virol. 67, 2365-2380. Schatz, D. G., Oettinger, M. A., and Baltimore, D. (1989). Cell 59, 1035-1048. Steitz, T. A. (1990). Quart. Rev. Biophys. 23, 205-280. Takahashi, M., Inaguma, Y., Hiai, H., and Hirose, E (1988). Mol. Cell. Biol. 8, 1853-1856. Thiem, S., and Miller, L. K. (1989). J. Virol. 63, 4489-4497.

A novel cysteine-rich sequence motif.

Cell, Vol. 64, 483-484, February8, 1991, Copyright© 1991 by Cell Press Letter to the Editor A Novel Cysteine-Rich Sequence Motif A number of protein...
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