.I. .Mol. Bid (lW7) 198. 31 l-326 Structure of a Complex of Catabolite Gene Activator Protein and Cyclic AMP Refined at 2-5 A Resolution Irene T. Weber ‘f C’enter for (‘hernictrl Physics X&ma1 Bureau of’ Standvrd.r Gaithersburg ND %0X%9. ~‘.S.A. and Thomas A. Steitz (Received 10 April 1.987. and in, rrviscd ,f’rorn 22 July 1987) Thr structure of A dimer of the Escherichin coli cataholite gene activator protein has been refined at 2.5 .A resolut,ion to a crystallographic* R-factor of .2@70,, starting with vo- ordinates Wed to the map at 1.9 At resolution. The two subunits are in different wnformations and each contains one bound molecule of the a.llost’eric activator. yvclica .1JlP. The amino-terminal domain is linked to thta smaller caarboxy-terminal domain ,h;- a nine-rrsidue hingr region that exists in different conformations in the two subunits. plvlng rise to approximat)ely a 30” rotation between the positions of the small domains relative to the larger domains. The amino-terminal dornain contains an antiparallel B-roll structure in which the interstrand hydrogen honding is well-determined. The P-roll can be descrihrd as a long antiparallel p-ribbon that folds into a right-handed supercoil and forms part of thcl cy-clic AMP binding site. Each q-&c AMP molecule is in an nr~ti conformation and has ionic and hydrogen bond interactions with both subunits. 1. Introduction The c~ataholite gene activator protein (CAP)$, also known as ca)-cnfica AMP receptor protein. rrgu- latrs tranwription of several catabolite-sensitive operons in Ewhrrichin wli (Zubay rt nl., 1970: I)e(‘rombrugghe & Pastan. 1978; T)eCrom hrugghe et al., 19X4). (‘ycliv AMP acts as an allosteric activat’or. Jn the presence of CAMP. (‘XI’ binds to spwific I)NA s~c~umc~s near the promoter region of operons suc~h as lactose. galavtose and maltose. CAP wtains onI>, a non-secluence-specific binding to l)SX in the absence of bound CAMP. (‘AT’ i.s a dimcar of 15.000 molecular weight with 209 amino acaid rrsidnes in each subunit, (Aiba et al.. I!W: (‘ossart 8 C~ic~quel-Sanzey, 1982). The crystals of (‘-41’ we gro\rw in the presence of 0.5 mM-cAMP at Io\v ionic- strrnpth (JlcKay Hr Fried. 1980). I’nder t Present address: Xational Cancer Institute. (‘rystallography Laboratory. BRI-FCRF. Frederick MU 21701. L1.S.A. $ Abbreviat,ions used: CAP, catabolite gene activator protein: c,4MP. cyclic AMP; m.i.r.. multiple isomorphous replacement,; r.m.s.. root-mean-square: 1 A = 0.1 nm. thew cwnditions (‘AT’ binds to the spwific 11X.4 seclwnws so w-e expect that the observed molecular strucature will be in t,he DNA binding wnformation. Thv crvstal structure of C’AP complexed with CAMP was init ially detrrrninrd at 2.9 L! resolution (MlrKa) & Stritz. 1981) and the sequence was fitted to t,he electron density (JlcKay ef ccl.. 198%). Each subunit of thr (‘Al’ dimer folds into two domains. The two subunits have different conformations: in the subunit in the ~~c~losed” c~onforrnation. the csarhoxy- terminal and amino-terminal domains lir c>loser toptli her than in the “open” conformation. Binding studies on the products of proteolytic digestion (Nilen c’t nl.. 1978: Aiba & KrakoLv. 1!381) suggest that there is a separate function for vac*h domain as is ohscrvt~tl for other gene regulatory prot,rins swh as I/r, wpressor (Platt et al.. 1973; Files & LYeher, 1976: Gislcr Kr il’rher. 1975) and larnhtia repressor (Pat,0 rf 01.. 1979). The larger amino-terminal tlorlrilin hinds a molecule of CAMP and shows homologies in amino acid sequence with thr rryulatory subunit of the mammalian rnxymr. (~=\MP-dependent protein kinase (W”rhrr fit al.. 1982b. 1987a). The smaller carboxy-terminal domains are involved in binding to T)N.-l and show