Sequence context modulates the stability of a GxxxG-mediated transmembrane helix-helix dimer

To quantify the relationship between sequence and transmembrane dimer stability, a systematic mutagenesis and thermodynamic study of the protein-protein interaction residues in the glycophorin A transmembrane helix-helix dimer was carried out. The results demonstrate that the glycophorin A transmembrane sequence dimerizes when its GxxxG motif is abolished by mutation to large aliphatic residues, suggesting that the sequence encodes an intrinsic propensity to self-associate independent of a GxxxG motif. In the presence of an intact GxxxG motif, the glycophorin A dimer stability can be modulated over a span of -0.5 kcal mol(-1) to +3.2 kcal mol(-1) by mutating the surrounding sequence context. Thus, these flanking residues play an active role in determining the transmembrane dimer stability. To assess the structural consequences of the thermodynamic effects of mutations, molecular models of mutant transmembrane domains were constructed, and a structure-based parameterization of the free energy change due to mutation was carried out. The changes in association free energy for glycophorin A mutants can be explained primarily by changes in packing interactions at the protein-protein interface. The energy cost of removing favorable van der Waals interactions was found to be 0.039 kcal mol(-1) per A2 of favorable occluded surface area. The value corresponds well with estimates for mutations in bacteriorhodopsin as well as for those mutations in the interiors of soluble proteins that create packing defects.     

Calculating the free energy of association of transmembrane helices

A large number of experimental studies have been devoted to quantifying the interaction between transmembrane (TM) helices in detergent micelles and, more recently, in bilayers. Theoretical calculation of association free energy of TM helices would be useful for predicting the propensity of given sequences to oligomerize and for understanding the difference between association in micelles and in bilayers. In this article, the theoretical foundation for calculating the standard association free energy of TM helices is laid out and is applied to glycophorin A in both micelles and bilayers. The standard association free energy is decomposed into the effective energy, translational, rotational, and conformational entropy terms. The effective energy of association is obtained by molecular dynamics simulations in an implicit membrane model. The translational and rotational entropy of association is calculated from the probability distribution of the translational and rotational degrees of freedom obtained from the molecular dynamics simulations. The side-chain conformational entropy of association is estimated from the probability distribution obtained by rigid rotation of all side-chain dihedral angles. The calculated standard association free energy of glycophorin A in N-dodecylphosphocholine micelles is in good agreement with the experimental value. The translational entropy cost is larger, whereas the rotational entropy cost is smaller in bilayers than in micelles. The standard association free energy in 1,2-dimyristoyl-sn-glycero-3-phosphocholine bilayers is calculated to be approximately 1.3 kcal/mol more favorable than in N-dodecylphosphocholine micelles, consistent with available experimental data.     

Standardizing the free energy change of transmembrane helix-helix interactions

Side-to-side associations of transmembrane alpha-helices are integral components of the structure and function of helical membrane proteins. A fundamental unknown in the understanding of the chemical principles driving the lateral interactions between transmembrane alpha-helices is the balance of forces arising from the polypeptide sequence versus the hydrophobic solvent. To begin to address this question, a consideration of basic thermodynamic principles has been applied to assess the experimental free energy change associated with transmembrane helix dimerization in micelles. This analysis demonstrates the ability to partition the apparent free energy of transmembrane helix-helix association into two components. The first component is a statistical energy term, which arises from the fact that there are an unequal number of reactants and products. The second component is a standard state free energy change, which informs on the molecular details of the transmembrane helix self-association reaction. The advantage of separating these two energy terms arises from the fact that extrapolation to the standard state free energy change normalizes the statistical energy term so that it applies equivalently in all experimental systems. Accompanying experimental results for the glycophorin A transmembrane alpha-helix dimer measured in micelles are well described by these theoretical components assuming an ideal-dilute solution.     

Study of the insulin dimerization: binding free energy calculations and per-residue free energy decomposition

A calculation of the binding free energy for the dimerization of insulin has been performed using the molecular mechanics-generalized Born surface area approach. The calculated absolute binding free energy is -11.9 kcal/mol, in approximate agreement with the experimental value of -7.2 kcal/mol. The results show that the dimerization is mainly due to nonpolar interactions. The role of the hydrogen bonds between the 2 monomers appears to give the direction of the interactions. A per-atom decomposition of the binding free energy has been performed to identify the residues contributing most to the self association free energy. Residues B24-B26 are found to make the largest favorable contributions to the dimerization. Other residues situated at the interface between the 2 monomers were found to make favorable but smaller contributions to the dimerization: Tyr B16, Val B12, and Pro B28, and to an even lesser extent, Gly B23. The energy decomposition on a per-residue basis is in agreement with experimental alanine scanning data. The results obtained from a single trajectory (i.e., the dimer trajectory is also used for the monomer analysis) and 2 trajectories (i.e., separate trajectories are used for the monomer and dimer) are similar.     

The stability of transmembrane helix interactions measured in a biological membrane

Despite some promising progress in the understanding of membrane protein folding and assembly, there is little experimental information regarding the thermodynamic stability of transmembrane helix interactions and even less on the stability of transmembrane helix-helix interactions in a biological membrane. Here we describe an approach that allows quantitative measurement of transmembrane helix interactions in a biological membrane, and calculation of changes in the interaction free energy resulting from substitution of single amino acids. Dimerization of several variants of the glycophorin A transmembrane domain are characterized and compared to the wild-type (wt) glycophorin A transmembrane helix dimerization. The calculated DeltaDeltaG(app) values are further compared with values found in the literature. In addition, we compare interactions between the wt glycophorin A transmembrane domain and helices in which critical glycine residues are replaced by alanine or serine, respectively. The data demonstrate that replacement of the glycine residues by serine is less destabilizing than replacement by alanine with a DeltaDeltaG(app) value of about 0.4 kcal/mol. Our study comprises the first measurement of a transmembrane helix interaction in a biological membrane, and we are optimistic that it can be further developed and applied.     

Detergents modulate dimerization, but not helicity, of the glycophorin A transmembrane domain.

Understanding how the lipid environment influences transmembrane helix association requires thermodynamic measurements that can be interpreted in terms of specific chemical interactions. We have used F?rster resonance energy transfer to measure dimerization of the glycophorin A transmembrane helix in detergent micelles. The observed Kd is at least two orders of magnitude weaker in sodium dodecyl sulfate than it is in zwitterionic detergents. In contrast, neither dimerization nor the detergent affects the secondary structure of the glycophorin A helix as measured by far-UV circular dichroism. These measurements support a long standing assumption about the glycophorin A transmembrane domain, that detergents uncouple helix formation from helix dimerization. The approach is applicable to a variety of systems in diverse environments, extending our ability to measure how interactions with complex solvents affect the thermodynamics of oligomerization.     

The transmembrane domains of ErbB receptors do not dimerize strongly in micelles

The epidermal growth factor receptors (erbB) constitute an important class of single pass transmembrane receptors involved in the transduction of signals important for cell proliferation and differentiation. Receptor association is a key event in the signal transduction process, but the molecular basis of this interaction is not fully understood. Previous biochemical and genetic studies have suggested that the single transmembrane helices of these receptor proteins might play a role in stabilizing the receptor complexes. To determine if the erbB transmembrane domains could provide a driving force to stabilize the receptor dimers, we carried out a thermodynamic study of these domains expressed as C-terminal fusion proteins with staphylococcal nuclease. Similar fusion constructs have been used successfully to investigate the oligomerization and association thermodynamics of a number of transmembrane sequences, including that of glycophorin A. Using SDS-PAGE analysis and sedimentation equilibrium analytical ultracentrifugation, we do not find strong, specific homo or hetero-interactions between the transmembrane domains of the erbB receptors in micellar solutions. Our results indicate that any preferential interactions between these domains in micellar solutions are extremely modest, of the order of 1 kcal mol(-1) or less. We applied a thermodynamic formalism to assess the effect of weakly interacting TM segments on the behavior of a covalently attached soluble domain. In the case of the ligand-bound EGFR ectodomain, we find that restriction of the ectodomain to the micellar phase by a hydrophobic TM, even in the absence of strong specific interactions, is largely sufficient to account for the previously reported increase in dimerization affinity.     

Towards a structural understanding of the smallest known oncoprotein: investigation of the bovine papillomavirus E5 protein using solution-state NMR

The homodimeric E5 protein from bovine papillomavirus activates the platelet-derived growth factor β receptor through transmembrane (TM) helix-helix interactions leading to uncontrolled cell growth. Detailed structural information for the E5 dimer is essential if we are to uncover its unique mechanism of action. In vivo mutagenesis has been used to identify residues in the TM domain critical for dimerization, and we previously reported that a truncated synthetic E5 peptide forms dimers via TM domain interactions. Here we extend this work with the first application of high-resolution solution-state NMR to the study of the E5 TM domain in SDS micelles. Using selectively 15N-labelled peptides, we first probe sample homogeneity revealing two predominate species, which we interpret to be monomer and dimer. The equilibrium between the two states is shown to be dependent on detergent concentration, revealed by intensity shifts between two sets of peaks in 15N-(1)H HSQC experiments, highlighting the importance of sample preparation when working with these types of proteins. This information is used to estimate a free energy of association (ΔGx°=-3.05 kcal mol(-1)) for the dimerization of E5 in SDS micelles. In addition, chemical shift changes have been observed that indicate a more pronounced change in chemical environment for those residues expected to be at the dimer interface in vivo versus those that are not. Thus we are able to demonstrate our in vitro dimer is comparable to that defined in vivo, validating the biological significance of our synthetic peptide and providing a solid foundation upon which to base further structural studies. Using detergent concentration to modulate oligomeric state and map interfacial residues by NMR could prove useful in the study of other homo-oligomeric transmembrane proteins.     

Association of a model transmembrane peptide containing gly in a heptad sequence motif

A peptide containing glycine at a and d positions of a heptad motif was synthesized to investigate the possibility that membrane-soluble peptides with a Gly-based, left-handed helical packing motif would associate. Based on analytical ultracentrifugation in C14-betaine detergent micelles, the peptide did associate in a monomer-dimer equilibrium, although the association constant was significantly less than that reported for the right-handed dimer of the glycophorin A transmembrane peptide in similar detergents. Fluorescence resonance energy transfer (FRET) experiments conducted on peptides labeled at their N-termini with either tetramethylrhodamine (TMR) or 7-nitrobenz-2-oxa-1,3-diazole (NBD) also indicated association. However, analysis of the FRET data using the usual assumption of complete quenching for NBD-TMR pairs in the dimer could not be quantitatively reconciled with the analytical ultracentrifugation-measured dimerization constant. This led us to develop a general treatment for the association of helices to either parallel or antiparallel structures of any aggregation state. Applying this treatment to the FRET data, constraining the dimerization constant to be within experimental uncertainty of that measured by analytical ultracentrifugation, we found the data could be well described by a monomer-dimer equilibrium with only partial quenching of the dimer, suggesting that the helices are most probably antiparallel. These results also suggest that a left-handed Gly heptad repeat motif can drive membrane helix association, but the affinity is likely to be less strong than the previously reported right-handed motif described for glycophorin A.     

Dimeric procaspase-3 unfolds via a four-state equilibrium process.

We have examined the folding and assembly of a catalytically inactive mutant of procaspase-3, a homodimeric protein that belongs to the caspase family of proteases. The caspase family, and especially caspase-3, is integral to apoptosis. The equilibrium unfolding data demonstrate a plateau between 3 and 5 M urea, consistent with an apparent three-state unfolding process. However, the midpoint of the second transition as well as the amplitude of the plateau are dependent on the protein concentration. Overall, the data are well described by a four-state equilibrium model in which the native dimer undergoes an isomeration to a dimeric intermediate, and the dimeric intermediate dissociates to a monomeric intermediate, which then unfolds. By fitting the four-state model to the experimental data, we have determined the free energy change for the first step of unfolding to be 8.3 +/- 1.3 kcal/mol. The free energy change for the dissociation of the dimeric folding intermediate to two monomeric intermediates is 10.5 +/- 1 kcal/mol. The third step in the unfolding mechanism represents the complete unfolding of the monomeric intermediate, with a free energy change of 7.0 +/- 0.5 kcal/mol. These results show two important points. First, dimerization of procaspase-3 occurs as a result of the association of two monomeric folding intermediates, demonstrating that procaspase-3 dimerization is a folding event. Second, the stability of the dimer contributes significantly to the conformational free energy of the protein (18.8 of 25.8 kcal/mol).     

Dimerization of the erythropoietin receptor transmembrane domain in micelles

Erythropoietin receptor (EpoR) homodimerization is an initial regulatory step in erythrocyte formation. Receptor dimers form before ligand binding, suggesting that association between receptor proteins is dependent on the receptor itself. EpoR dimerization is an essential step in erythropoiesis, and misregulation of this dimerization has been implicated in several disease states, including multi-lineage leukemias; nevertheless, how EpoR regulates its own dimerization is unclear. In vivo experiments suggest the single-pass transmembrane helix is the strongest candidate for driving ligand-independent association. To address the self-association potential of this transmembrane segment, we studied its interaction energetics in micelles by utilizing a previously successful Staphylococcal nuclease (SN-EpoR TM) fusion protein. This fusion protein strategy allows expression of the EpoR transmembrane domain in Escherichia coli independent of the other EpoR domains. Sedimentation equilibrium analytical ultracentrifugation of the detergent-solubilized SN-EpoR TM demonstrated that the murine EpoR transmembrane domain self-associates to form dimers. Although this interaction is not as stable as the dimerization of the well-studied glycophorin A transmembrane dimer, the murine EpoR transmembrane domain dimer is more stable than the interactions of the colon carcinoma kinase 4 transmembrane domain. The same experiments with the human EpoR transmembrane domain, which differs from the mouse sequence by only three residues, revealed a less favorable interaction than that of the murine sequence and is only slightly more favorable than that expected for non-preferential binding. These results suggest that the mouse and human receptor proteins may differ in the roles they play in signaling.     

Comparison of binding of mixed ribose-deoxyribose analogues of CUCU to a ribozyme and to GGAGAA by equilibrium dialysis: evidence for ribozyme specific interactions with 2' OH groups

Dissociation constants at 15 degrees C were measured by equilibrium dialysis for the binding of rCrUrCrU, dCrUrCrU, rCdUrCrU, rCrUdCrU, and rCrUrCdU to the L-21 ScaI form of the self-splicing group I LSU intron from Tetrahymena thermophila. Substitution of deoxyribose for ribose in each of the middle two positions makes the free energy change for binding 1-2 kcal/mol less favorable, compared to about 0.3 kcal/mol less favorable for each of the terminal positions. Dissociation constants for binding of the same oligomers to rGGAGAA were measured by optical melting methods. Substitution of a single deoxyribose for ribose makes the free energy change for binding less favorable by 0.4-0.9 kcal/mol for this simple duplex formation. Comparison of the effects for binding to ribozyme and to rGGAGAA indicate that ribozyme-specific tertiary interactions dependent on the middle two 2' OH groups of rCrUrCrU add about 2 kcal/mol of favorable free energy for binding to L-21 ScaI. Comparisons are made with results from gel retardation studies [Pyle, A. M., McSwiggen, J. A., & Cech, T. R. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 8187-8191; Pyle, A. M., & Cech, T. R. (1991) Nature (London) 350, 628-631].     

Estimation of helix-helix association free energy from partial unfolding of bacterioopsin

To obtain thermodynamic information about interactions between transmembrane helices in integral membrane proteins, partial unfolding of bacterioopsin in ethanol/water mixtures was studied by F?rster-type resonance energy transfer (FRET) from tryptophan to a dansyl group on Lys 41. Tryptophan to dansyl FRET was detected by measuring sensitized emission at 490-500 nm from 285 nm excitation. FRET was observed in dansylbacterioopsin in apomembranes and in detergent micelles but not in 90% ethanol/water or in the chymotrypsin fragment C2 (residues 1-71). The main fluorescence donors are Trp 86 and Trp 182. Increase of FRET from C2 with added chymotrypsin fragment C1 (residues 72-248) provides an estimate of the C1-C2 association constant as 7.7 x 10(6) M(-1). With increasing ethanol concentration, the FRET signal from dansylbacterioopsin in detergent micelles disappeared with a sharp transition above 60% ethanol. No transition occurred in Trp fluorescence from bacterioopsin lacking the dansyl acceptor, nor did dansyl model compounds undergo a similar transition. Light scattering measurements show that the detergent micelles dissipate below 50% ethanol. Thus the observed transition is likely to be a partial unfolding of bacterioopsin. Assuming a two-state unfolding model, the free energy of unfolding was obtained by extrapolation as 9.0 kcal/mol. The slope of the transition (m-value) was -0.8 kcal mol(-1) M(-1). The unfolding process probably involves dissociation of several helices. The rate of association was measured by stopped-flow fluorometry. Two first-order kinetic processes were observed, having approximately equal weights, with rate constants of 2.32 s (-1) and 0.185 s(-1).     

Changes in apparent free energy of helix-helix dimerization in a biological membrane due to point mutations

We present an implementation of the TOXCAT membrane protein self-association assay that measures the change in apparent free energy of transmembrane helix dimerization caused by point mutations. Quantifying the reporter gene expression from cells carrying wild-type and mutant constructs shows that single point mutations that disrupt dimerization of the transmembrane domain of glycophorin A reproducibly lower the TOXCAT signal more than 100-fold. Replicate cultures can show up to threefold changes in the level of expression of the membrane bound fusion construct, and correcting for these variations improves the precision of the calculated apparent free energy change. The remarkably good agreement between our TOXCAT apparent free energy scale and free energy differences from sedimentation equilibrium studies for point mutants of the glycophorin A transmembrane domain dimer indicate that sequence changes usually affect membrane helix-helix interactions quite similarly in these two very different environments. However, the effects of point mutations at threonine 87 suggest that intermonomer polar contacts by this side-chain contribute significantly to dimer stability in membranes but not in detergents. Our findings demonstrate that a comparison of quantitative measurements of helix-helix interactions in biological membranes and genuine thermodynamic data from biophysical measurements on purified proteins can elucidate how changes in the lipidic environment modulate membrane protein stability.     

The thermodynamics of bovine and porcine insulin and proinsulin association determined by concentration difference spectroscopy

Difference spectroscopy was used to determine the equilibrium constants and thermodynamic parameters for the monomer-dimer association of bovine and porcine insulin and bovine proinsulin at pH 2.0 and 7.0. At pH 2 delta G degree 25, delta S degree, and delta H degree for dimerization of bovine insulin were found to be -6.6 kcal/mol, -18 cal/mol-deg, and -12 kcal/mol, respectively. Porcine insulin behaved similarly to bovine insulin in its dimerization properties in that delta G degree 25, delta S degree, and delta H degree were found to be -6.8 kcal/mol, -14 cal/mol-deg, and -11 kcal/mol, respectively. At pH 7 delta G degree 25, delta S degree, and delta H degree for dimerization of bovine insulin were found to be -7.2 kcal/mol, -16 cal/mol/deg, and -12 kcal/mol, respectively. At pH 7.0 delta G degree 25, delta S degree, and delta H degree for dimerization of porcine insulin were -6.7 kcal/mol, -11.6 cal/mol-deg, and -10 kcal/mol, respectively. The similarity in the thermodynamic parameters of both insulin species at the different pH's suggests that there are minimal structural changes at the monomer-monomer contact site over this pH range. The dimerization of both insulin species is under enthalpic control. This may suggest that the formation of the insulin dimer is not driven by hydrophobic bonding but, rather, is driven by the formation between subunits of four hydrogen bonds in an apolar environment. At pH 2 delta G degree 25, delta S degree, and delta H degree for dimerization of bovine proinsulin were found to be -5.3 kcal/mol, -26 cal/mol-deg, and -13 kcal/mol, respectively. At pH 7 delta G degree 25, delta S degree, and delta H degree for dimerization of proinsulin were -5.9 kcal/mol, -4.2 cal/mol-deg, and -7.2 kcal/mol, respectively. Although the presence of the C-peptide on proinsulin does not drastically affect the overall free energy change of dimer formation (as compared to insulin), the other thermodynamic parameters are rather drastically altered. This may be because of electrostatic interactions of groups on the C-peptide with groups on the B-chain which are near the subunit contact site in the insulin dimer.     

Effect of detergents on the association of the glycophorin a transmembrane helix

We have examined the role of the environment on the interactions between transmembrane helices using, as a model system, the dimerization of the glycophorin A transmembrane helix. In this study we have focused on micellar environments and have examined a series of detergents that include a range of alkyl chain lengths, combined with ionic, zwitterionic, and nonionic headgroups. For each we have measured how the apparent equilibrium constant depends on the detergent concentration. In two detergents we also measured the thermal sensitivity of the equilibrium constant, from which we derive the van't Hoff enthalpy and entropy. We show that several simple models are inadequate for explaining our results; however, models that include the effect of detergent concentration on detergent binding are able to account for our measurements. Our analysis suggests that the effects of detergents on helix association are due to a pair of opposing effects: an enthalpic effect, which drives association as the detergent concentration is increased and which is sensitive to the chemical nature of the detergent headgroup, opposed by an entropic effect, which drives peptide dissociation as the detergent concentration is raised. Our results also indicate that the monomer-monomer interface is relatively hydrophilic and that association within detergent micelles is driven by the enthalpy change. The wide variations in glycophorin a dimmer, stability with the detergent used, together with the realization that this results from the balance between two opposing effects, suggests that detergents might be selected that drive association rather than dissociation of peptide dimers.     

Kinetic and thermodynamic study of the tetramerization equilibrium of phosphorylase b

The association equilibrium of phosphorylase b, induced by AMP and in the presence of Mg2+, has been shown to be a reversible process that follows second order and first order reversible rate laws in the direction of tetramerization and dimerization respectively, this fact being independent of temperature and of enzyme and AMP concentrations. Moreover, rate and equilibrium constants have been evaluated and their dependence on temperature and AMP concentration studied in this work. An important role that the existence of two classes of AMP binding sites per enzymatic subunit plays in the aggregation properties of the enzyme has also been emphasized. In the presence of 0.1 and 1 mM AMP (binding to the high affinity site), the values of the change in enthalpy, activation energy of dimerization and activation energy of tetramerization are: -36 kcal/mol, +36 kcal/mol, and 0 kcal/mol respectively. Binding of AMP to the low affinity site (10 mM AMP) yields significant changes in the self-association equilibrium, since the preceding parameters reach the following values: -18, +32, and +14 kcal/mol.     

The contribution of C alpha-H...O hydrogen bonds to membrane protein stability depends on the position of the amide

Structural analyses of membrane proteins reveal a large number of C(alpha)-H...O contacts between transmembrane helices, presumed to be hydrogen bonds. Recent experiments produced conflicting results for the contribution of such hydrogen bonds to membrane protein stability. An FTIR study estimated an energy of -0.88 kcal/mol for the G79-C(alpha)-H...I76-O hydrogen bond in glycophorin A, whereas a mutagenesis study showed that the A51-C(alpha)-H...T24-O(gamma) hydrogen bond does not stabilize bacteriorhodopsin. Here, we reconcile these results using molecular mechanics calculations and an implicit membrane model (IMM1). With explicit hydrogen atoms, the potential energy of the G79-C(alpha)-H...I76-O interaction in GpA ranges from -0.54 to -0.9 kcal/mol and its contribution to stability (effective energy) from -0.49 to -0.83 kcal/mol, depending on the structural model used. The average values of these quantities in GpA-like motifs are similar. In bR, the corresponding numbers for the A51-C(alpha)-H...T24-O(gamma) interaction are +0.15 and +0.32 kcal/mol. The difference results from the different arrangement of the interacting groups and specifically the position of the acceptor with respect to the C(alpha) and N atoms. This conclusion likely applies to soluble proteins as well.     

Total chemical synthesis of the integral membrane protein influenza A virus M2: role of its C-terminal domain in tetramer assembly.

The M2 protein from influenza A virus is a 97-residue homotetrameric membrane protein that functions as a proton channel. To determine the features required for the assembly of this protein into its native tetrameric state, the protein was prepared by total synthesis using native chemical ligation of unprotected peptide segments. Circular dichroism spectroscopy of synthetic M2 protein in dodecylphosphocholine (DPC) micelles indicated that approximately 40 residues were in an alpha-helical secondary structure. The tetramerization of the full-length protein was compared to that of a 25-residue transmembrane (TM) fragment. Analytical ultracentrifugation demonstrated that both the peptide and the full-length protein in DPC micelles existed in a monomer-tetramer equilibrium. Comparison of the association constants for the two sequences showed the free energy of tetramerization of the full-length protein was more favorable by approximately 7 kcal/mol. Partial proteolysis of DPC-solubilized M2 was used as a further probe of the structure of the full-length protein. A 15-20-residue segment C-terminal to the membrane-spanning region was found to be highly resistant to digestion by chymotrypsin and trypsin. This region, which we have modeled as an extension of the TM helices, may help to stabilize the tetrameric assembly.     

The GxxxG-containing transmembrane domain of the CCK4 oncogene does not encode preferential self-interactions

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.