Eph receptor tyrosine kinases and their membrane-associated ligands, the ephrins, are essential regulators of axon guidance, cell migration, segmentation, and angiogenesis. There are two classes of vertebrate ephrin ligands which have distinct binding specificities for their cognate receptors. Multimerization of the ligands is required for receptor activation, and ephrin ligands themselves signal intracellularly upon binding Eph receptors. We have determined the structure of the extracellular domain of mouse ephrin-B2. The ephrin ectodomain is an eight-stranded beta barrel with topological similarity to plant nodulins and phytocyanins. Based on the structure, we have identified potential surface determinants of Eph/ephrin binding specificity and a ligand dimerization region. The high sequence similarity among ephrin ectodomains indicates that all ephrins may be modeled upon the ephrin-B2 structure presented here.

The ubiquitin conjugating (E2) enzyme encoded by CDC34 (UBC3) in Saccharomyces cerevisiae is required for the G1 to S transition of the cell cycle. CDC34 consists of a 170 residue amino‐terminal domain that is homologous to that found in other E2s, followed by a 125 residue carboxyl‐terminal domain that is specific to CDC34. We found that a truncation mutant of CDC34 which lacked the CDC34 carboxyl‐terminal domain could not support the essential function of CDC34 in the cell cycle in vivo. To explore further the role of the carboxyl‐terminal domain in determining the cell cycle function of CDC34, we constructed and characterized genes encoding chimeric E2s incorporating sequences from CDC34 and the related but functionally distinct E2 RAD6 (UBC2). We found that a construct encoding a chimeric RAD6‐CDC34 ubiquitin conjugating enzyme, in which the 21 residue acidic carboxyl‐terminal domain of RAD6 has been replaced with the 125 residue carboxyl‐terminal domain of CDC34, performed the essential functions of CDC34 in vivo. This chimeric E2 also complemented the growth deficiency, UV sensitivity and sporulation deficiency of rad6 mutant strains. Deletion analysis of the CDC34 carboxyl‐terminal domain in both CDC34 and the RAD6‐CDC34 chimeric E2 identified a region comprising residues 171–244 of CDC34 that was sufficient to confer CDC34 function on the amino‐terminal domains of CDC34 and RAD6. We suggest that this region interacts with substrates of CDC34 or with trans‐acting factors (such as CDC34‐specific ubiquitin protein ligases) that govern the substrate selectivity of CDC34. Congruent results demonstrating a positive role for the carboxyl‐terminal domain of CDC34 in the essential function of CDC34 have also been obtained by Silver et al. (1992) and are reported in the accompanying paper.

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Formaldehyde crosslinking has been widely used to study binding of specific proteins to DNA elements in intact cells. However, previous studies have not determined if this crosslinker preserves the bona fide pattern of DNA binding. Here we show that formaldehyde crosslinking of Drosophila embryos maps an interaction of the transcription factor Zeste to a known target element in the Ultrabithorax promoter. This data agrees broadly with previous mapping of the same Zeste binding sites by in vivo UV crosslinking, though the formaldehyde method does give a low, possibly artifactual signal on other DNA fragments that is not detected by the UV method. We also demonstrate, using an in vitro assay, that formaldehyde crosslinking accurately reflects the DNA binding specificities of both Zeste and a second transcription factor, Eve. The crosslinking reagent methylene blue is shown to preserve DNA binding specificity in vitro as well. Our results suggest that crosslinking by formaldehyde, and possibly also by methylene blue, provide an accurate guide to the interaction of proteins with their high affinity target sites in cells.

GrpE is the nucleotide exchange factor for the Escherichia coli molecular chaperone DnaK, the bacterial homologue of Hsp70. In the temperature range of the bacterial heat shock response, the long helices of GrpE undergo a helix-to-coil transition, and GrpE exhibits non-Arrhenius behavior with respect to its nucleotide exchange function. It is hypothesized that GrpE acts as a thermosensor and that unwinding of the long helices of E. coli GrpE reduces its activity as a nucleotide exchange factor. In turn, it was proposed that temperature-dependent down-regulation of the activity of GrpE may increase the time in which DnaK binds its substrates at higher temperatures. A combination of thermodynamic and hydrodynamic techniques, in concert with the luciferase refolding assay, were used to characterize a molecular mechanism in which the long helices of GrpE are thermodynamically linked with the beta-domains via an intramolecular contact between Phe86 and Arg183. These "thermosensing" long helices were found to be necessary for full activity as a nucleotide exchange factor in the luciferase refolding assay. Point mutations in the beta-domains and in the long helices of GrpE destabilized the beta-domains. Engineered disulfide bonds in the long helices alternately stabilized the long helices and the four-helix bundle. This allowed the previously reported 75 degrees C thermal transition seen in the excess heat capacity function as monitored by differential scanning calorimetry to be further characterized. The observed thermal transition represents the unfolding of the four-helix bundle and the beta-domains. The thermal transitions for these two domains are superimposed but are not thermodynamically linked.