pubs.acs.org/Biochemistry Published on Web 08/14/2009 r 2009 American Chemical Society Biochemistry 2009, 48, 8559–8567 8559 DOI: 10.1021/bi900534r N-Terminal Aliphatic Residues Dictate the Structure, Stability, Assembly, and Small Molecule Binding of the Coiled-Coil Region of Cartilage Oligomeric Matrix Protein † Susheel K. Gunasekar, ‡ Mukta Asnani, ‡ Chandani Limbad, ‡ Jennifer S. Haghpanah, ‡ Wendy Hom, ‡ Hanna Barra, ‡ Soumya Nanda, ‡ Min Lu, § and Jin Kim Montclare* ,‡, ) ‡ Department of Chemical and Biological Sciences, Polytechnic Institute of New York University, Brooklyn, New York 11201, § Department of Biochemistry, Weill Medical College of Cornell University, New York, New York 10021, and ) Department of Biochemistry, SUNY-Downstate Medical Center, Brooklyn, New York 11203 Received March 29, 2009; Revised Manuscript Received July 19, 2009 ABSTRACT: The coiled-coil domain of cartilage oligomeric matrix protein (COMPcc) assembles into a homopentamer that naturally recognizes the small molecule 1,25-dihydroxyvitamin D 3 (vit D). To identify the residues critical for the structure, stability, oligomerization, and binding to vit D as well as two other small molecules, all-trans-retinol (ATR) and curcumin (CCM), here we perform an alanine scanning mutagenesis study. Ten residues lining the hydrophobic pocket of COMPcc were mutated into alanine; of the mutated residues, the N-terminal aliphatic residues L37, L44, V47, and L51 are responsible for maintaining the structure and function. Furthermore, two polar residues, T40 and Q54, within the N-terminal region when converted into alanine improve the R-helical structure, stability, and self-assembly behavior. Helical stability, oligomerization, and binding appear to be linked in a manner in which mutations that abolish helical structure and assembly bind poorly to vit D, ATR, and CCM. These results provide not only insight into COMPcc and its functional role but also useful guidelines for the design of stable, pentameric coiled-coils capable of selectively storing and delivering various small molecules. Cartilage oligomeric matrix protein (COMP) is a noncolla- genic glycoprotein present in cartilage, tendons, ligaments, and osteoblasts (1-4). Belonging to the family of thromospondins (TSPs), 1 COMP has a pentameric boquetlike structure with a molecular mass of 524 kDa (1, 5). It is composed of an N- terminal globular domain followed by an epidermal growth factor (EGF) type 2 repeat domain, a calcium binding type 3 repeat domain, and a C-terminal globular domain (5, 6). The C- terminal globular domain of COMP interacts with collagen I and II and induces the formation of collagen fibrils (7, 8). Mutations in COMP result in genetic disorders, including pseudoachon- droplasia and multiple epiphyseal dysplasia in humans charac- terized by short stature and other vertebral abnormalities (9-14). Although COMP is comprised of various domains, it is assembled into a homopentamer via an N-terminal coiled-coil domain (COMPcc). COMPcc possesses a hydrophobic pore that is 7.3 nm long with a diameter of 0.2-0.6 nm (15-18) (Figure 1). Recent structural studies reveal that the hormone 1,25-dihydrox- yvitamin D 3 (vit D) can bind in the pore (17). This, in addition to other biochemical studies, suggests a putative role of the penta- meric coiled coil in storing vit D for signaling events during morphogenesis and repair of cartilage and bone (19). Further- more, COMPcc has the ability to bind hydrophobic molecules like all-trans-retinol (ATR), retinoic acid, elaidic acid, cyclohex- ane, and benzene as demonstrated by an increased thermal stability (17). This ability to house a variety of small molecules in the pore of the protein represents an important feature for storage and delivery. To date, mutagenesis of the COMPcc domain has been centered on the Q54 residue since on the basis of crystallographic studies, it appears to separate the hydrophobic pore into two compartments (17, 20). Mutation of Q54 into leucine resulted in higher stability (>120 °C) compared to that of COMPcc (108 °C) (20). A slightly different Q54I mutant bound ATR with a dissociation equilibrium constant (K D ) of 0.8 mM, exhibiting an affinity similar to that of the COMPcc (K D = 0.6 mM) (17). Although these studies in addition to the structure provide insight into the significance of the single Q54 residue, there are a set of residues that line the pocket of COMPcc that may contribute to its overall structure and function that have not yet been explored. Here we perform a single-alanine mutagenesis study to identify the a and d residues within the hydrophobic pore of COMPcc critical for the structure, stability, oligomerization, and binding to the target vit D as well as ATR and curcumin (CCM) (Figure 1). Residues within the N-terminal region extending to Q54 play a significant role in the structure and self-assembly of † This work was supported by the Air Force Office of Scientific Research YIP (FA-9550-07-1-0060) and DURIP (FA-9550-08-1-0266) (J.K.M.), in part by the National Science Foundation MRSEC Program through Grant DMR-0820341 and GK-12 Fellows Grant 0741714 (J.S. H.), by National Institutes of Health Grant AI42382 (M.L.), by a Wechsler Award (J.K.M.), by Unilever (J.K.M.), and by the Othmer Institute for Interdisciplinary Studies (J.K.M.). *To whom correspondence should be addressed: Department of Chemical and Biological Sciences, Polytechnic Institute of New York University, Brooklyn, NY 11201. Telephone: (718) 260-3679. Fax: (718) 260-3676. E-mail: jmontcla@poly.edu. 1 Abbreviations: COMPcc, cartilage oligomeric matrix protein coiled coil; TSP, thromospondin; EGF, epidermal growth factor; vit D, 1,25- dihydroxyvitamin D 3 ; ATR, all-trans-retinol; CCM, curcumin; LB, Luria broth; IPTG, isopropyl thio-β-galactopyranoside; BSA, bovine serum albumin; PBS, phosphate-buffered saline; BS 3 , bis- (sulfosuccinimidyl) suberate; TFA, trifluoroacetic acid; DMSO, di- methyl sulfoxide; MALDI, matrix-assisted laser desorption ionization; CD, circular dichroism; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Downloaded by NEW YORK UNIV on September 8, 2009 | http://pubs.acs.org Publication Date (Web): August 14, 2009 | doi: 10.1021/bi900534r