ASEdb: a database of alanine mutations and their effects on the free energy of binding in protein interactions.

The Alanine Scanning Energetics database (ASEdb) is a searchable database of single alanine mutations in protein-protein, protein-nucleic acid, and protein-small molecule interactions for which binding affinities have been experimentally determined. In cases where structures are available, it contains surface areas of the mutated side chain and links to the PDB entries. It is useful for studying the contribution of single amino acids to the energetics of protein interactions, and can be updated by researchers as new data are generated.

Analysis of protein dimerization and ligand binding of orphan receptor HNF4alpha.

Hepatocyte nuclear factor 4alpha (HNF4alpha) (NR2A1), an orphan member of the nuclear receptor superfamily, binds DNA exclusively as a homodimer even though it is very similar in amino acid sequence to retinoid X receptor alpha (RXRalpha), which heterodimerizes readily with other receptors. Here, experimental analysis of residues involved in protein dimerization and studies on a reported ligand for HNF4alpha are combined with a structural model of the HNF4alpha ligand-binding domain (LBD) (residues 137 to 384). When K300 (in helix 9) and E327 (in helix 10) of HNF4alpha1 were converted to the analogous residues in RXRalpha (E390 and K417, respectively) the resulting construct did not heterodimerize with the wild-type HNF4alpha, although it was still able to form homodimers and bind DNA. Furthermore, the double mutant did not heterodimerize with RXR or RAR but was still able to dimerize in solution with an HNF4alpha construct truncated at amino acid residue 268. This suggests that the charge compatibility between helices 9 and 10 is necessary, but not sufficient, to determine dimerization partners, and that additional residues in the HNF4alpha LBD are also important in dimerization. The structural model of the HNF4alpha LBD and an amino acid sequence alignment of helices 9 and 10 in various HNF4 and other receptor genes indicates that a K(X)(26)E motif can be used to identify HNF4 genes from other organisms and that a (E/D(X)(26-29)K/R) motif can be used to predict heterodimerization of many, but not all, receptors with RXR. In vitro analysis of another HNF4alpha mutant construct indicates that helix 10 also plays a structural role in the conformational integrity of HNF4alpha. The structural model and experimental analysis indicate that fatty acyl CoA thioesters, the proposed HNF4alpha ligands, are not good candidates for a traditional ligand for HNF4alpha. Finally, these results provide insight into the mechanism of action of naturally occurring mutations in the human HNF4alpha gene found in patients with maturity onset diabetes of the young 1 (MODY1).

Co-evolution of proteins with their interaction partners.

The divergent evolution of proteins in cellular signaling pathways requires ligands and their receptors to co-evolve, creating new pathways when a new receptor is activated by a new ligand. However, information about the evolution of binding specificity in ligand-receptor systems is difficult to glean from sequences alone. We have used phosphoglycerate kinase (PGK), an enzyme that forms its active site between its two domains, to develop a standard for measuring the co-evolution of interacting proteins. The N-terminal and C-terminal domains of PGK form the active site at their interface and are covalently linked. Therefore, they must have co-evolved to preserve enzyme function. By building two phylogenetic trees from multiple sequence alignments of each of the two domains of PGK, we have calculated a correlation coefficient for the two trees that quantifies the co-evolution of the two domains. The correlation coefficient for the trees of the two domains of PGK is 0. 79, which establishes an upper bound for the co-evolution of a protein domain with its binding partner. The analysis is extended to ligands and their receptors, using the chemokines as a model. We show that the correlation between the chemokine ligand and receptor trees' distances is 0.57. The chemokine family of protein ligands and their G-protein coupled receptors have co-evolved so that each subgroup of chemokine ligands has a matching subgroup of chemokine receptors. The matching subfamilies of ligands and their receptors create a framework within which the ligands of orphan chemokine receptors can be more easily determined. This approach can be applied to a variety of ligand and receptor systems.

Phosphatidylinositol transfer protein (PITPalpha) stimulates in vitro intra-Golgi transport.

Using a cell-free assay designed to reconstitute cis-to-medial intra-Golgi vesicular transport, we identified at least four crude activities in bovine brain cytosol that stimulate this assay. We have purified one of these activities to near homogeneity and have identified this Mr 36 kDa protein to be the alpha isoform of phosphatidylinositol transfer protein (PITPalpha) by N-terminal peptide sequencing, immunoreactivity with PITP-specific antisera, and the ability of recombinant PITPalpha to stimulate in vitro intra-Golgi transport. From these data, we conclude that in vitro Golgi transport is facilitated by PITPalpha.

Anatomy of hot spots in protein interfaces.

Binding of one protein to another is involved in nearly all biological functions, yet the principles governing the interaction of proteins are not fully understood. To analyze the contributions of individual amino acid residues in protein-protein binding we have compiled a database of 2325 alanine mutants for which the change in free energy of binding upon mutation to alanine has been measured (available at http://motorhead. ucsf.edu/thorn/hotspot). Our analysis shows that at the level of side-chains there is little correlation between buried surface area and free energy of binding. We find that the free energy of binding is not evenly distributed across interfaces; instead, there are hot spots of binding energy made up of a small subset of residues in the dimer interface. These hot spots are enriched in tryptophan, tyrosine and arginine, and are surrounded by energetically less important residues that most likely serve to occlude bulk solvent from the hot spot. Occlusion of solvent is found to be a necessary condition for highly energetic interactions.