Dimeric proteins can arise by the swapping of structural domains between monomers. The prevalence of this occurrence is unknown. Ribonuclease A (RNase A) is assumed to be a monomer near physiological conditions. Here, this hypothesis is tested and found to be imprecise. The two histidine residues (His12 and His119) in the active site of RNase A arise from two domains (S-peptide and S-protein) of the protein. The H12A and H119A variants have 105-fold less ribonucleolytic activity than does the wild-type enzyme. Incubating a 1:1 mixture of the H12A and H119A variants at pH 6.5 and 65 °C results in a 103-fold increase in ribonucleolytic activity. A large quantity of active dimer can be produced by lyophilizing a 1:1 mixture of the H12A and H119A variants from acetic acid. At pH 6.5 and 65 °C, the ribonucleolytic activity of this dimer converges to that of the dimer formed by simply incubating the monomers, as expected for a monomer–dimer equilibrium. The equilibrium dissociation constant for the dimer is near 2 mM at both 65 and 37 °C. This value of Kd is only 20-fold greater than the concentration of RNase A in the cow pancreas, suggesting that RNase A dimers exist in vivo. The intrinsic ability of RNase A to form dimers under physiological conditions is consistent with a detailed model for the evolution of homodimeric proteins. Dimers of “monomeric” proteins could be more prevalent than is usually appreciated.

The three-dimensional (3D) structure of Corynebacterium glutamicum diaminopimelate d-dehydrogenase in a ternary complex with NADPH and l-2-amino-6-methylene-pimelate has been solved and refined to a resolution of 2.1 Å. l-2-Amino-6-methylene-pimelate was recently synthesized and shown to be a potent competitive inhibitor (5 μM) vs. meso-diaminopimelate of the Bacillus sphaericus dehydrogenase (Sutherland et al., 1999). Diaminopimelate dehydrogenase catalyzes the reversible NADP+-dependent oxidation of the d-amino acid stereocenter of meso-diaminopimelate, and is the only enzyme known to catalyze the oxidative deamination of a d-amino acid. The enzyme is involved in the biosynthesis of meso-diaminopimelate and l-lysine from l-aspartate, a biosynthetic pathway of considerable interest because it is essential for growth of certain bacteria. The dehydrogenase is found in a limited number of species of bacteria, as opposed to the alternative succinylase and acetylase pathways that are widely distributed in bacteria and plants. The structure of the ternary complex reported here provides a structural rationale for the nature and potency of the inhibition exhibited by the unsaturated l-2-amino-6-methylene-pimelate against the dehydrogenase. In particular, we compare the present structure with other structures containing either bound substrate, meso-diaminopimelate, or a conformationally restricted isoxazoline inhibitor. We have identified a significant interaction between the α-l-amino group of the unsaturated inhibitor and the indole ring of Trp144 that may account for the tight binding of this inhibitor.

The high accuracy of X-ray analyses at atomic resolution is now able to display subtle deformations from standard geometry of building blocks in proteins. From the analysis of nine ultra-high resolution protein structures, we derived the first experimental evidence that a significant pyramidalization at the main-chain carbonyl carbon atom occurs in proteins. Our findings also show that this pyramidalization is related to the main-chain ψ torsion angle. The carbonyl carbon atoms of residues that adopt αR and extended conformations show a clear preference for positive and negative pyramidalization, respectively. The agreement between our data and those previously obtained from small molecule structures demonstrates that carbon pyramidalization is an intrinsic property of the peptide structure. Although small in magnitude, the pyramidalization is well preserved in the complex folded state of a macromolecular structure that results from the interplay of many different forces. In addition, this property of the peptide group may have interesting implications for the enzymatic reactions involving the carbonyl carbon atoms.

The EF-hand calcium-binding protein S100B has been shown to interact in vitro in a calcium-sensitive manner with many substrates. These potential S100B target proteins have been screened for the preservation of a previously identified consensus sequence across species. The results were compared to known structural and in vitro properties of the proteins to rationalize choices for potential binding partners. Our approach uncovered four oligomeric proteins tubulin (α and β), glial fibrillary acidic protein (GFAP), desmin, and vimentin that have conserved regions matching the consensus sequence. In the type III intermediate filament proteins (GFAP, vimentin, and desmin), this region corresponds to a portion of a coiled-coil (helix 2A), the structural element responsible for their assembly. In tubulin, the sequence matches correspond to regions of α and β tubulin found at the αβ tubulin interface. In both cases, these consensus sequence matches provide a logical explanation for in vitro observations that S100B is able to inhibit oligomerization of these proteins.

A sequence variant of human MIP-1α, in which Asp26 has been replaced by Ala, has been chemically modified by the addition of 13C-labeled methyl groups at each of the lysine residues and the N-terminus. The sites of methylation have been verified by a combination of MALDI-TOF mass spectrometric experiments and tryptic digestion followed by N-terminal mapping. The effect of the modification on the structure and activity of the protein have been determined by analytical ultra-centrifugation, 13C NMR spectroscopy and receptor binding studies. The results of these experiments suggest that huMIP-1α D26A (BB10010), when present as a dimer, adopts a globular structure, like MCP-3, rather than the elongated or cylindrical structure determined for dimers of huMIP-1β and RANTES.

In the Acknowledgments section that was published in the above reference article (Volume 9, Number 7, 2000, pp. 1282–1293), the wrong PDB ID number was used. The correct ID number is 1F5Y.