title: The GxxxG-containing transmembrane domain of the CCK4 oncogene does not encode preferential self-interactions.
abstract: 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.
authors.affiliation: T. C. Jenkins Department of Biophysics, Johns Hopkins University, 3400 North Charles Street, Baltimore, Maryland 21218, USA.
authors.forename: Felix J
authors.initials: FJ
authors.lastname: Kobus
authors.forename: Karen G
authors.initials: KG
authors.lastname: Fleming
mesh_terms: D020816:Amino Acid Motifs / Q000235:genetics; D000595:Amino Acid Sequence; D015815:Cell Adhesion Molecules / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary / Q000235:genetics; D020794:Receptor Protein-Tyrosine Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013816:Thermodynamics; D014461:Ultracentrifugation
chemical_list: D015815:Cell Adhesion Molecules; D008565:Membrane Proteins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; C096905:PTK7 protein, human; D020794:Receptor Protein-Tyrosine Kinases