Structure, Vol. 11, 1303–1307, October, 2003, 2003 Elsevier Science Ltd. All rights reserved. DOI 10.1016/j.str.2003.09.004A Closed Compact Structureof Native Ca2ⴙ-Calmodulinacetoalanine is well defined (Figure 1A), as is the otherposttranslational modification (trimethyllysine 115); infact, all atoms of calmodulin are present in density ex-Jennifer L. Fallon1and Florante A. Quiocho1,2,*1Howard Hughes Medical Institute and2Verna Marrs McLean Department of Biochemistryand Molecular Biology cept for some atoms at the ends of Lys 13 and Lys21. We are not aware of another structure of a nativeBaylor College of MedicineHouston, Texas 77030 eukaryotic Ca2⫹-CaM in which both posttranslationalmodifications are clearly resolved.As shown in Figure 1B, the overall structure is verycompact and globular, the two domains (N- and C-)coming together in cis orientation such that helices I andSummaryII of the N-domain and helices VII and V (respectively) ofthe C-domain are very close together, essentially closingCalmodulin has been a subject of intense scrutinyoff the channel where ␣-helical MLCK-type target do-since its discovery because of its unusual propertiesmains or small molecule cyclic drugs would normallyin regulating the functions of about 100 diverse targetbind. The linker in this structure assumes a clearly de-enzymes and structural proteins. The original and tofined type 1 reverse turn involving residues 78, 79, 80,date only crystal conformation of native eukaryoticand 81. These residues are the same ones implicatedCa2ⴙ-calmodulin (Ca2ⴙ-CaM) is a very extended mole-in linker flexibility in a solution NMR study (Barbato etcule with two widely separated globular domainsal., 1992) and also have very high temperature factorslinked by an exposed long helix. Here we report thein crystal structures of the extended form (e.g., see Babu1.7 A˚X-ray structure of a new native Ca2ⴙ-CaM thatet al., 1985; Chattopadhyaya et al., 1992; Wilson andis in a compact ellipsoidal conformation and shows aBrunger, 2000).sharp bend in the linker helix and a more contractedThe space between the two domains in the “interior”N-terminal domain. This conformation may offer advan-of the protein appears to contain solvent molecules only.tages for recognition of kinase-type calmodulin targetsTwo small clumps of density in this region have eachor small organic molecule drugs.been modeled as a three water cluster, since the proteinwas treated with EGTA to remove bound Ca2⫹and dia-lysed exhaustively, thereby dislodging any potentialIntroductionbound endogenous ligand, and reconstituted with cal-cium prior to crystallization (Experimental Procedures).Although much is now known about the three-dimen-The interdomain contacts that stabilize this compactsional structures of Ca2⫹-CaM with bound target do-structure consist of sixteen hydrogen bonds (⬍3.4 A˚)mains or small organic drug molecules (Clapperton etand twenty-four van der Waals interactions (⬍4A˚), inal., 2002; Drum et al., 2002; Ikura et al., 1992; Kurokawaaddition to a number of polar-nonpolar contacts (Figureet al., 2001; Meador et al., 1992, 1993, 1995; Schumacher1B). Significant regions of hydrogen bonding occur be-et al., 2001; Vandonselaar et al., 1994), the only unboundtween the N-terminal helix I and the final EF hand helicesnative crystal structures of Ca2⫹-CaM currently availableVII and VIII in the C-domain, as well as between the twoall resemble the first structure solved 18 years ago (Babuhelices of the collapsed linker (IV and V, see Figure 1A).et al., 1985). This structure revealed two domains, eachOther hydrogen bonds involve residues at the ends ofwith a pair of bound Ca2⫹metals, separated by a longthe loops in each domain (Gln 41 and Gln 114). Theexposed helix (the linker helix), and is thus referred tononpolar binding regions are almost exactly the same,as the extended form of Ca2⫹-CaM since most of thewith slightly over half involving helix I interactions astarget bound structures are more compact. The 1.7 A˚above. These helix I interdomain contacts seal off thisresolution structure of the unbound native brain Ca2⫹-end of the “binding channel” along with the large changeCaM reported here reveals a unique new compact form,in the N-domain conformation discussed below. Mostwhich adds considerable new insight into the conforma-of the residues involved in the interdomain contacts intional extremes available to this most unusual regulatorythis structure are involved in target peptide contacts inprotein molecule.the CaMKII and MLCK target-bound structures (Meadoret al., 1993).The architecture of the first Ca2⫹-CaM structure isResults and Discussionrelatively simple, with two domains (in trans orientation)each composed of two Ca2⫹binding helix-loop-helix EFThe structure of the brain Ca2⫹-CaM was determined byhands joined by a short loop (Babu et al., 1985). Thesingle isomorphous replacement from a Pb derivativelast helix of the first domain and the first helix of theand refined at 1.7 A˚resolution to an R-factor of 0.188,second domain together form the very unusual exposedR-free of 0.235, and good geometry. In the present Ca2⫹-long central helix generally referred to as the linker,CaM structure, the electron density of the N-terminalwhose middle portion is capable of expanding into asmaller or larger loop as required for optimum domainpositioning during target recognition (Meador et al.,*Correspondence: [email protected] 1. Unbound Compact Structure ofNative Ca2⫹-CaM(A) Stereoview of the electron density (2Fo-Fc at 1 ␴ level) for the N-domain of Ca2⫹-CaM,showing the N-terminal acetoalanine (n-ACE)and residues 2, 3, and 4 as a ball and stickmodel. Carbon atoms are green, oxygens arered, and nitrogens are blue.(B) Stereoview of the ribbon diagram of Ca2⫹-CaM showing interdomain contacts. The CaMbackbone is purple, loops are brown, and cal-ciums are blue spheres. The sidechains thatmake contact (including 16 hydrogen bonds(⬍3.4 A˚) and 24 van der Waals interactions(⬍4A˚)) between domains are shown (atomcolors as Figure 1A, with sulfur yellow). Heli-ces are numbered from I-VIII.1993, 1995; Meador and Quiocho, 2002). Within each (top much closer to base) for the new structure, withN-domain interhelical angle values that are approxi-domain for Ca2⫹-CaM, the first and forth helices can beseen as forming the base of a large hydrophobic pocket, mately halfway between those for