Altering dimerization specificity by changes in surface electrostatics.

Arc repressor forms a homodimer in which the subunits intertwine to create a single globular domain. To obtain Arc sequences that fold preferentially as heterodimers, variants with surface patches of excess positive or negative charge were designed. Several but not all oppositely charged sequence pairs showed preferential heterodimer formation. In the most successful design pair, alpha helix B of one subunit contained glutamic acids at positions 43, 46, 47, 48, and 50, whereas the other subunit contained lysines or arginines at these positions. A continuum electrostatic model captures many features of the experimental results and suggests that the most successful designs include elements of both positive and negative design.

Structure of a transiently phosphorylated switch in bacterial signal transduction.

Receiver domains are the dominant molecular switches in bacterial signalling. Although several structures of non-phosphorylated receiver domains have been reported, a detailed structural understanding of the activation arising from phosphorylation has been impeded by the very short half-lives of the aspartylphosphate linkages. Here we present the first structure of a receiver domain in its active state, the phosphorylated receiver domain of the bacterial enhancer-binding protein NtrC (nitrogen regulatory protein C). Nuclear magnetic resonance spectra were taken during steady-state autophosphorylation/dephosphorylation, and three-dimensional spectra from multiple samples were combined. Phosphorylation induces a large conformational change involving a displacement of beta-strands 4 and 5 and alpha-helices 3 and 4 away from the active site, a register shift and an axial rotation in helix 4. This creates an exposed hydrophobic surface that is likely to transmit the signal to the transcriptional activation domain.

Structural and functional analyses of activating amino acid substitutions in the receiver domain of NtrC: evidence for an activating surface.

The bacterial enhancer-binding protein NtrC activates transcription when phosphorylated on aspartate 54 in its amino (N)-terminal regulatory domain or when altered by constitutively activating amino acid substitutions. The N-terminal domain of NtrC, which acts positively on the remainder of the protein, is homologous to a large family of signal transduction domains called receiver domains. Phosphorylation of an aspartate residue in a receiver domain modulates the function of a downstream target, but the accompanying structural changes are not clear. In the present work we examine structural and functional differences between the wild-type receiver domain of NtrC and mutant forms carrying constitutively activating substitutions. Combinations of such substitutions resulted in both increased structural changes in the N-terminal domain, monitored by NMR chemical shift differences, and increased transcriptional activation by the full-length protein. Structural changes caused by substitutions outside the active site (D86N and A89T) were not only local but extended over a substantial portion of the N-terminal domain including the region from alpha-helix 3 to beta-strand 5 ("3445 face") and propagating to the active site. Interestingly, the activating substitution of glutamate for aspartate at the site of phosphorylation (D54E) also triggered structural changes in the 3445 face. Thus, the active site and the 3445 face appear to interact. Implications with respect to how phosphorylation may affect the structure of receiver domains and how structural changes may be communicated to the remainder of NtrC are discussed.

Three-dimensional solution structure of the N-terminal receiver domain of NTRC.

NTRC is a transcriptional enhancer binding protein whose N-terminal domain is a member of the family of receiver domains of two-component regulatory systems. Using 3D and 4D NMR spectroscopy, we have completed the 1H, 15N, and 13C assignments and determined the solution structure of the N-terminal receiver domain of the NTRC protein. Determination of the three-dimensional structure was carried out with the program X-PLOR (Brünger, 1992) using a total of 915 NMR-derived distance and dihedral angle restraints. The resultant family of structures has an average root mean square deviation of 0.81 A from the average structure for the backbone atoms involved in well-defined secondary structure. The structure is comprised of five alpha-helices and a five-stranded parallel beta-sheet, in a (beta/alpha)5 topology. Comparison of the solution structure of the NTRC receiver domain with the crystal structures of the homologous protein CheY in both the Mg(2+)-free and Mg(2+)-bound forms [Stock, A.M., Mottonen, J. M., Stock, J. B., & Schutt, C. E. (1989) Nature 337, 745-749; Volz, K., & Matsumura, P. (1991) J. Biol. Chem. 296, 15511-15519; Stock, A. M., Martinez-Hackert, E., Rasmussen, B. F., West, A. H., Stock, J. B., Ringe, D., & Petsko, G. A. (1993) Biochemistry 32, 13375-13380; Bellsolell, L., Prieto, J., Serrano, L., & Coll, M. (1994) J. Mol. Biol. 238, 489-495] reveals a very similar fold, with the only significant difference occurring in the positioning of helix 4 relative to the rest of the protein. Examination of the conformation of consensus residues of the receiver domain superfamily [Volz, K. (1993) Biochemistry 32, 11741-11753] in the structures of the NTRC receiver domain and CheY establishes the structural importance of residues whose side chains are involved in hydrogen bonding or hydrophobic core interactions. The importance of some nonconsensus residues which may be conserved for their ability to fulfill helix capping roles is also discussed.

The E1 protein of bovine papilloma virus 1 is an ATP-dependent DNA helicase.

For efficient DNA replication of papillomaviruses, only two viral-encoded proteins, E1 and E2, are required. Other proteins and factors are provided by the host cell. E2 is an enhancer of both transcription and replication and is known to help E1 bind cooperatively to the origin of DNA replication. E1 is sufficient for replication in extracts prepared from permissive cells, but the activity is enhanced by E2. Here we show that purified E1 can act as an ATP-dependent DNA helicase. To measure this activity, we have used strand displacement, unwinding of topologically constrained DNA, denaturation of duplex fragments, and electron microscopy. The ability of E1 to unwind circular DNA is found to be independent of origin-specific viral DNA sequences under a variety of experimental conditions. In unfractionated cellular extracts, E1-dependent viral DNA replication is origin-dependent, but at elevated E1 concentrations, replication can occur on non-origin-containing DNA templates. This conversion from an origin-dependent replication system to a nonspecific initiator system is discussed in the context of the current understanding of the initiation of chromosomal DNA replication.