Computation and mutagenesis suggest a right-handed structure for the synaptobrevin transmembrane dimer.

Biological membrane fusion involves a highly precise and ordered set of protein-protein interactions. Synaptobrevin is a key player in this process. Mutagenesis studies of its single transmembrane segment suggest that it dimerizes in a sequence specific manner. Using the computational methods developed for the successful structure prediction of the glycophorin A transmembrane dimer, we have calculated a structural model for the synaptobrevin dimer. Our computational search yields a well-populated cluster of right-handed structures consistent with the experimentally determined dimerization motif. The three-dimensional structure contains an interface formed primarily by leucine and isoleucine side-chain atoms and has no interhelical hydrogen bonds. The model is the first three-dimensional picture of the synaptobrevin transmembrane dimer and provides a basis for further focused experimentation on its structure and association thermodynamics.

Specificity in transmembrane helix-helix interactions can define a hierarchy of stability for sequence variants.

The folding, stability, and oligomerization of helical membrane proteins depend in part on a precise set of packing interactions between transmembrane helices. To understand the energetic principles of these helix-helix interactions, we have used alanine-scanning mutagenesis and sedimentation equilibrium analytical ultracentrifugation to quantitatively examine the sequence dependence of the glycophorin A transmembrane helix dimerization. In all cases, we found that mutations to alanine at interface positions cost free energy of association. In contrast, mutations to alanine away from the dimer interface showed free energies of association that are insignificantly different from wild-type or are slightly stabilizing. Our study further revealed that the energy of association is not evenly distributed across the interface, but that there are several "hot spots" for interaction including both glycines participating in a GxxxG motif. Inspection of the NMR structure indicates that simple principles of protein-protein interactions can explain the changes in energy that are observed. A comparison of the dimer stability between different hydrophobic environments suggested that the hierarchy of stability for sequence variants is conserved. Together, these findings imply that the protein-protein interaction portion of the overall association energy may be separable from the contributions arising from protein-lipid and lipid-lipid energy terms. This idea is a conceptual simplification of the membrane protein folding problem and has implications for prediction and design.

Quaternary and domain structure of glycoprotein processing glucosidase II.

Glucose trimming from newly synthesized glycoproteins regulates their interaction with the calnexin/calreticulin chaperone system. We have recently proposed that glucosidase II consisted of two different subunits, alpha and beta. The alpha subunit is the catalytic component, and deletion of its homologue in yeast obliterates glucosidase II activity. Deletion of the homologue of the noncatalytic beta subunit in Schizosaccharomices pombe drastically reduces glucosidase II activity, but the role of the beta subunit in glucosidase II activity has not been established. Furthermore, a direct interaction between alpha and beta subunits has not been demonstrated. Using chemical cross-linking and hydrodynamic analysis by analytical ultracentrifugation, we found that the two subunits form a defined complex, composed of one catalytic subunit and one accessory subunit (alpha(1)beta(1)) with a molecular mass of 161 kDa. The complex had an s value of 6.3 S, indicative of a highly nonglobular shape. The asymmetric shape of the alpha(1)beta(1) complex was confirmed by its high susceptibility to proteases. The beta subunit could be proteolytically removed from the alpha(1)beta(1) complex without affecting catalysis, demonstrating that it is not required for glucosidase II activity in vitro. Furthermore, we isolated a monomeric C-terminal fragment of the alpha subunit, which retained full glucosidase activity. We conclude that the catalytic core of glucosidase II resides in a globular domain of the alpha subunit, which can function independently of the beta subunit, while the complete alpha and beta subunits assemble in a defined heterodimeric complex with a highly extended conformation, which may favor interaction with other proteins in the endoplasmic reticulum (ER). Through its C-terminal HDEL signal, the beta subunit may retain the complete alpha(1)beta(1) complex in the ER.

Human 5-aminoimidazole-4-carboxamide ribonucleotide transformylase/inosine 5'-monophosphate cyclohydrolase. A bifunctional protein requiring dimerization for transformylase activity but not for cyclohydrolase activity.

The bifunctional enzyme aminoimidazole carboxamide ribonucleotide transformylase/inosine monophosphate cyclohydrolase (ATIC) is responsible for catalysis of the last two steps in the de novo purine pathway. Gel filtration studies performed on human enzyme suggested that this enzyme is monomeric in solution. However, cross-linking studies performed on both yeast and avian ATIC indicated that this enzyme might be dimeric. To determine the oligomeric state of this protein in solution, we carried out sedimentation equilibrium analysis of ATIC over a broad concentration range. We find that ATIC participates in a monomer/dimer equilibrium with a dissociation constant of 240 +/- 50 nM at 4 degrees C. To determine whether the presence of substrates affects the monomer/dimer equilibrium, further ultracentrifugation studies were performed. These showed that the equilibrium is only significantly shifted in the presence of both AICAR and a folate analog, resulting in a 10-fold reduction in the dissociation constant. The enzyme concentration dependence on each of the catalytic activities was studied in steady state kinetic experiments. These indicated that the transformylase activity requires dimerization whereas the cyclohydrolase activity only slightly prefers the dimeric form over the monomeric form.

Probing stability of helical transmembrane proteins.

Sedimentation equilibrium in the analytical ultracentrifuge provides membrane biochemists with a tool to quantitatively probe thermodynamics of associating systems in detergent environments. As long as conditions of reversibility are met, the free energy of interaction can be measured in varied hydrophobic environments, pH values, ionic strengths, and temperatures. Although the absolute value of the interaction free energy of membrane protein subunits will no doubt depend on the hydrophobic environment, experiments in any one environment will allow subunit associations to be placed on a relative scale of interaction. The temperature dependence of the free energy change may provide more thorough information about the thermodynamics of helix-helix association in micelles.

Riding the wave: structural and energetic principles of helical membrane proteins.

Genome sequencing efforts have revealed that perhaps as many as 20-40% of open reading frames in complex organisms may encode proteins containing at least one helical transmembrane segment. Contrasting with this approaching tidal wave of helical membrane proteins is the fact that our understanding of the sequence-structure-function relationships for membrane proteins lags far behind that of soluble proteins. This looming reality emphasizes the tremendous biochemical and structural work that remains to be done on helical membrane proteins in order to elucidate the structural and energetic principles that specify and stabilize their folds, which define their functions. These facts are not lost on the pharmaceutical industry, where successful therapeutics and major discovery efforts are targeting membrane proteins.

Oligomerization of a MutS mismatch repair protein from Thermus aquaticus.

The MutS DNA mismatch protein recognizes heteroduplex DNAs containing mispaired or unpaired bases. We have examined the oligomerization of a MutS protein from Thermus aquaticus that binds to heteroduplex DNAs at elevated temperatures. Analytical gel filtration, cross-linking of MutS protein with disuccinimidyl suberate, light scattering, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry establish that the Taq protein is largely a dimer in free solution. Analytical equilibrium sedimentation showed that the oligomerization of Taq MutS involves a dimer-tetramer equilibrium in which dimer predominates at concentrations below 10 microM. The DeltaG(0)(2-4) for the dimer to tetramer transition is approximately -6.9 +/- 0.1 kcal/mol of tetramer. Analytical gel filtration of native complexes and gel mobility shift assays of an maltose-binding protein-MutS fusion protein bound to a short, 37-base pair heteroduplex DNA reveal that the protein binds to DNA as a dimer with no change in oligomerization upon DNA binding.

Visual arrestin activity may be regulated by self-association.

Visual arrestin is the protein responsible for rapid quenching of G-protein-coupled receptor signaling. Arrestin exists as a latent inhibitor which must be 'activated' upon contact with a phosphorylated receptor. X-ray crystal structures of visual arrestin exhibit a tetrameric arrangement wherein an asymmetric dimer with an extensive interface between conformationally different subunits is related to a second asymmetric dimer by a local two-fold rotation axis. To test the biological relevance of this molecular organization in solution, we carried out a sedimentation equilibrium analysis of arrestin at both crystallographic and physiological protein concentrations. While the tetrameric form can exist at the high concentrations used in crystallography experiments, we find that arrestin participates in a monomer/dimer equilibrium at concentrations more likely to be physiologically relevant. Solution interaction analysis of a proteolytically modified, constitutively active form of arrestin shows diminished dimerization. We propose that self-association of arrestin may provide a mechanism for regulation of arrestin activity by (i) ensuring an adequate supply for rapid quenching of the visual signal and (ii) limiting the availability of active monomeric species, thereby preventing inappropriate signal termination.

A revised model for the oligomeric state of the N-ethylmaleimide-sensitive fusion protein, NSF.

The N-ethylmaleimide-sensitive fusion protein (NSF) is an ATPase that plays an essential role in intracellular membrane trafficking. Previous reports have concluded that NSF forms either a tetramer or a trimer in solution, and that assembly of the oligomer is essential for efficient activity in membrane transport reactions. However, in recent electron microscopic analyses NSF appears as a hexagonal cylinder similar in size to related ATPases known to be hexamers. We have therefore reevaluated NSF's oligomeric state using a variety of quantitative biophysical techniques. Sedimentation equilibrium and sedimentation velocity analytical ultracentrifugation, transmission electron microscopy with rotational image analysis, scanning transmission electron microscopy, and multiangle light scattering all demonstrate that, in the presence of nucleotide, NSF is predominantly a hexamer. Sedimentation equilibrium results further suggest that the NSF hexamer is held together by oligomerization of its D2 domains. The sedimentation coefficient, s20,w0, of 13.4 (+/-0. 1) S indicates that NSF has unusual hydrodynamic characteristics that cannot be solely explained by its shape. The demonstration that NSF is a hexameric oligomer highlights structural similarities between it and several related ATPases which act by switching the conformational states of their protein substrates in order to activate them for subsequent reactions.

The effects of salt on the TATA binding protein-DNA interaction from a hyperthermophilic archaeon.

This study investigates the thermodynamics of the interaction of the TATA box binding protein (TBP) from Pyrococcus woesei (Pw) with an oligonucleotide containing a specific binding site. Pw is a hyperthermophilic archeal organism which exists under conditions of high salt and high temperature. A measurable protein-DNA interaction only occurs at high salt concentrations. Isothermal titration calorimetric binding studies were performed under a range of salts (potassium chloride, potassium phosphate, potassium acetate and sodium acetate) at varying concentrations (0.8 to 1.6 M). At the high salt concentrations used the observed equilibrium binding constant increases with increasing salt concentration. This is very different to the effect reported for all other protein-DNA interactions which have been studied at lower salt concentrations. Thermodynamic data suggest that the protein-DNA interaction at high salt concentration is accompanied by the removal of large numbers of water molecules from the buried hydrophobic surface area. In addition, the involvement of ions appears to influence the binding which can be explained by binding of cations in the interface between the electrostatically negative lateral lobes on the protein and the negatively charged DNA.

The effect of point mutations on the free energy of transmembrane alpha-helix dimerization.

Glycophorin A forms homodimers through interaction of the single, helical transmembrane domains of the monomers. The dimers are stable in sodium dodecylsulfate (SDS), permitting a number of studies that have identified a critical motif of residues that mediates dimer formation. We have used analytical ultracentrifugation to measure the energy of dimerization in a non-denaturing detergent solution and have observed the changes in energy arising from two of the mutants previously studied. Use of the detergent pentaoxyethylene octyl ether (C8E5) is a great advantage, since its micelles are neutrally buoyant and the detergent allows a reversible association to occur between monomer and dimer states of the glycophorin A transmembrane helices during the time-scale of sedimentation equilibrium. Use of this detergent in analytical ultracentrifugation may enable a wide range of studies of molecular association events in membrane proteins. We find that the glycophorin A transmembrane helix dimerizes with a dissociation constant of 240(+/-50) nM, corresponding to a free energy of dissociation of 9.0(+/-0.1) kcal mol-1. Point mutants that were found to be disruptive in SDS (L75A, I76A) reduced the dimer affinity in the C8E5 detergent environment (Kd=1.7(+/-0.2) microM and 4.2(+/-0.9) microM, respectively). Thus, the earlier findings are placed on a quantitative, relative energy scale of association by our measurements. Molecular modeling and simulations suggest that the energy differences can be accounted for as changes in van der Waals interactions between helices.

What makes a protein a protein? Hydrophobic core designs that specify stability and structural properties.

Here we describe how the systematic redesign of a protein's hydrophobic core alters its structure and stability. We have repacked the hydrophobic core of the four-helix-bundle protein, Rop, with altered packing patterns and various side chain shapes and sizes. Several designs reproduce the structure and native-like properties of the wild-type, while increasing the thermal stability. Other designs, either with similar sizes but different shapes, or with decreased sizes of the packing residues, destabilize the protein. Finally, overpacking the core with the larger side chains causes a loss of native-like structure. These results allow us to further define the roles of tight residue packing and the burial of hydrophobic surface area in the construction of native-like proteins.

A zinc-binding domain involved in the dimerization of RAG1.

Recombination-activating gene 1 (RAG1), as well as RAG2, are the only lymphoid-specific genes required for V(D)J recombination. RAG1 protein contains a C3HC4 zinc-binding motif (zinc ring finger) that binds two zinc ions. We have found that RAG1 contains additional zinc-binding motifs in the form of two separate C2H2 zinc finger sequences. One of the zinc fingers, in combination with the C3HC4 subdomain, forms a highly specific dimerization domain. A combination of biophysical techniques has been used to determine the energetics of association, the overall shape of the dimerization domain, and the relative orientation of the monomeric subunits within the dimer. These results provide direct evidence that a C3HC4 motif is involved in a protein-protein interaction, in this case via homodimer formation. In addition, the observation that the dimerization domain includes multi-class zinc binding motifs, namely both a zinc finger and a C3HC4 subdomain, has important implications for other C3HC4-containing proteins. The position of this dimerization domain in the N-terminal third of the RAG1 sequence of 1040 amino acid residues may have a significant influence on the activities associated with the C-terminal domains of the protein.