Award Number: DAMD17-02-1-0427 TITLE: Energetics and structure prediction of the network of homo- and hetero- Oligomers Formed by the Transmembrane Domains of the ErbBReceptor Family of Proteins

The recently cloned colon carcinoma kinase 4 (CCK4) oncogene contains an evolutionarily conserved GxxxG motif in its single transmembrane domain (TMD). It has previously been suggested that this pairwise glycine motif may provide a strong driving force for transmembrane helix -helix interactions. Since CCK4 is thought to represent a new member of the receptor tyrosine kinase family, interactions between the TMDs may be important in receptor self-association and activation of signal transduction pathways. To determine whether this conserved CCK4 TMD can drive protein -protein interactions, we have carried out a thermodynamic study using the TMD expressed as a Staphylococcal nuclease (SN) fusion protein. Similar SN -TMD fusion proteins have been used to determine the sequence specificity and thermodynamics of transmembrane helix -helix interactions in a number of membrane proteins, including glycophorin A. Using sedimentation equilibrium in C14 betaine micelles, we discovered that the CCK4 TMD is unable to drive strong protein -protein interactions. At high protein/detergent ratios, the SN-CCK4 fusion protein will dimerize, but a stochastic model for protein association in micelles can explain the observed dimer population. For low-affinity interactions such as the one studied here, an understanding of this discrete stochastic distribution of membrane proteins in micelles is important for distinguishing between preferential and random self-interactions, which can both influence the oligomeric population. The lack of a thermodynamically meaningful self-association propensity for the CCK4 TMDs demonstrates that a GxxxG motif is not sufficient to drive transmembrane helix -helix interactions. Receptor tyrosine kinases (RTKs) 1 are known to play an important role in the development and/or progression of many forms of cancer. A molecular understanding of the mechanisms by which these receptors promote cellular transformation is critical for the understanding and treatment of human cancer. In normal cells, RTKs are transmembrane signaling proteins that transmit biological signals from the extracellular environment to the interior of the cell. The regulated transmission of these signals is important for many cellular events, such as cell growth and differentiation, axonal growth, epithelial growth and development ( 1). RTKs are misregulated in many human cancers including breast cancer (erbB2/Her2) ( 2, 3), ovarian cancer (erbB2) ( 2, 3), melanoma (CCK4) (4), and colon cancer (erbB1 and CCK4) ( 5, 6). The predominant RTK-related genetic alteration in human cancer is not mutation of the receptor protein, but rather it is amplification of the gene. Subsequent overexpression of receptor proteins leads to constitutive stimulation of the RTK activity and uncontrolled cellular signaling. Recently, colon carcinoma kinase 4 (CCK4), a new member of the receptor tyrosine kinase superfamily was cloned from human colon carcinoma-derived cell lines ( 7). The CCK4 mRNA for this gene was variably expressed in colon carcinoma derived cell lines but not expressed in human adult colon tissues. In contrast, it is expressed in fetal colon of the mouse ( 7). These observations suggest that the normal role for CCK4 may be in development of the colon followed by down regulation in the adult tissue. The unusual expression of CCK4 in colon carcinoma cell lines might indicate that the protein is functioning abnormally in adult colon cells, which suggests a role in colon carcinoma development and/or proliferation. Orthologues for CCK4 have been cloned from other species ( 8-10). Amino acid residues within the transmembrane domain (TMD) are among the conserved features ( 9). The CCK4 TMD is 55% identical to the transmembrane sequences of the chicken Klg and HydraLemonorthologues (9), and a pattern of residues with helical periodicity is conserved. Within the conserved residues of the TMD is a GxxxG sequence, a motif thought to promote interactions between transmembrane helices ( 11, 12). The evolutionary conservation of the CCK4 transmembrane sequence suggests the hypothesis that it may play a functionally important role in signaling that involves transmembrane helix -helix interactions. To test whether this CCK4 TMD may serve as a driving force for protein-protein interactions, we carried out a thermodynamic study of the TMD using sedimentation equilibrium in detergent micelles. † This work was supported by a grant from the NIH (GM57534) and by a Career Award from the Department of Defense (DAMD1702-1-0427). * To whom correspondence should be addressed. Voice: 410-5167256. Fax: 410-516-4118. E-mail: Karen.Fleming@jhu.edu 1 Abbreviations: CCK4, colon carcinoma kinase 4; C14 betaine, 3-(N,N-dimethylmyristyl-ammonio)propanesulfonate; RTK, receptor tyrosine kinases; SN, Staphylococcal nuclease; TMD, transmembrane domain; GpA, glycophorin A; SN-CCK4, a fusion protein composed of SN and the transmembrane domain of CCK4; SN -GpA, a fusion protein composed of SN and the transmembrane domain of glycophorin A. 1464 Biochemistry2005,44, 1464-1470 10.1021/bi048076l CCC: $30.25 © 2005 American Chemical Society Published on Web 01/08/2005 EXPERIMENTAL METHODS Cloning and Sample Preparation. An image clone for the CCK4 protein was purchased from Invitrogen. The sequence encoding the transmembrane region was amplified using the polymerase chain reaction using primers containing unique SmaI and BamH I sites, and the PCR product was cloned C-terminal toStaphylococcalnuclease (SN) in an IPTGinducible pET11a expression construct from which the open reading frame for the glycophorin A transmembrane had been excised. The DNA sequence was verified by double-stranded DNA sequencing. The Cys-to-Ser mutation (immediately before the KKR) was introduced using the QuickChange mutagenesis kit. The SN -CCK4s and SN-CCK4xs proteins were purified using the published protocol for the SN -GpA proteins ( 13). Immediately before sedimentation equilibrium analysis, samples were exchanged by ion-exchange chromatography into buffer containing 10 mM C14 betaine. SigmaUltra grade C14 betaine was purchased from Sigma. Sedimentation Equilibrium Analytical Ultracentrifugation. Sedimentation equilibrium experiments were performed at 25°C using a Beckman XL-A analytical ultracentrifuge using six-sector cells equipped with quartz windows in an AnTi 60 rotor as described previously in detail ( 13-15). The samples were centrifuged for lengths of time sufficient to achieve equilibrium determined by using the MATCH algorithm. Equilibrium data obtained from absorbance at 280 nm were analyzed by nonlinear least-squares curve fitting of radial concentration profiles with the Macintosh version of NONLIN (16) using the equations describing the reversible association in sedimentation equilibrium. For each global fit, nine data sets were used consisting of three different initial protein concentrations (initial A280 absorbance values of 0.9, 0.6, and 0.3 cm-1) analyzed at three significantly different speeds (20000, 24500, 30000 revolutions per minute). The monomeric molecular masses and partial specific volumes were calculated using the program SEDNTERP ( 17), and these parameters were held constant in fitting the absorbance versus radius profiles. The equilibrium constant was converted from absorbance to (bulk molar) units using molar extinction coefficients of 20952 and 21425 cm -1 M-1 for the SN-CCK4s and SN-CCK4xs proteins, respectively. These values were calculated by adjusting the experimental molar extinction coefficient for SN for the additional mass of the TMD of interest as has been previously done for the glycophorin A transmembrane domain ( 18). The apparent free energy of association was calculated from the apparent monomer -dimer equilibrium constant in the standard way, ∆G° App ) RT ln KApp. The standard state mole fraction free energy of association was calculated using the ideal-dilute assumption as ∆G° x ) RT ln Kx whereKx ) KApp[micellarC14]w and [micellarC14] w is the concentration of micellar C14 betaine expressed on the aqueous molar scale ( 14, 15). Sequence Motifs.Sequence motifs were identified using the online TMSTAT calculation ( 11) (http:// bioinfo.mbb.yale.edu/tmstat/) with the extended version of the CCK4 TMD.