Analysis of the costructure of the simian virus 40 T-antigen origin binding domain with site I reveals a correlation between GAGGC spacing and spiral assembly.

Polyomavirus origins of replication contain multiple occurrences of G(A/G)GGC, the high-affinity binding element for the viral initiator T-antigen (T-ag). The site I regulatory region of simian virus 40, involved in the repression of transcription and the enhancement of DNA replication initiation, contains two GAGGC sequences arranged head to tail and separated by a 7-bp AT-rich sequence. We have solved a 3.2-Å costructure of the SV40 origin-binding domain (OBD) bound to site I. We have also established that T-ag assembly on site I is limited to the formation of a single hexamer. These observations have enabled an analysis of the role(s) of the OBDs bound to the site I pentanucleotides in hexamer formation. Of interest, they reveal a correlation between the OBDs bound to site I and a pair of OBD subunits in the previously described hexameric spiral structure. Based on these findings, we propose that spiral assembly is promoted by pentanucleotide pairs arranged in a head-to-tail manner. Finally, the possibility that spiral assembly by OBD subunits accounts for the heterogeneous distribution of pentanucleotides found in the origins of replication of polyomaviruses is discussed.

Asymmetric assembly of Merkel cell polyomavirus large T-antigen origin binding domains at the viral origin.

The double-stranded DNA polyomavirus Merkel cell polyomavirus (MCV) causes Merkel cell carcinoma, an aggressive but rare human skin cancer that most often affects immunosuppressed and elderly persons. As in other polyomaviruses, the large T-antigen of MCV recognizes the viral origin of replication by binding repeating G(A/G)GGC pentamers. The spacing, number, orientation, and necessity of repeats for viral replication differ, however, from other family members such as SV40 and murine polyomavirus. We report here the 2.9 Å crystal structure of the MCV large T-antigen origin binding domain (OBD) in complex with a DNA fragment from the MCV origin of replication. Consistent with replication data showing that three of the G(A/G)GGC-like binding sites near the center of the origin are required for replication, the crystal structure contains three copies of the OBD. This stoichiometry was verified using isothermal titration calorimetry. The affinity for G(A/G)GGC-containing double-stranded DNA was found to be ~740 nM, approximately 8-fold weaker than the equivalent domain in SV40 for the analogous region of the SV40 origin. The difference in affinity is partially attributable to DNA-binding residue Lys331 (Arg154 in SV40). In contrast to SV40, a small protein-protein interface is observed between MCV OBDs when bound to the central region of the origin. This protein-protein interface is reminiscent of that seen in bovine papilloma virus E1 protein. Mutational analysis indicates, however, that this interface contributes little to DNA binding energy.

Mutational analysis of the energetics of the GrpE.DnaK binding interface: equilibrium association constants by sedimentation velocity analytical ultracentrifugation.

DnaK, the prokaryotic Hsp70 molecular chaperone, requires the nucleotide exchange factor and heat shock protein GrpE to release ADP. GrpE and DnaK are tightly associated molecules with an extensive protein-protein interface, and in the absence of ADP, the dissociation constant for GrpE and DnaK is in the low nanomolar range. GrpE reduces the affinity of DnaK for ADP, and the reciprocal linkage is also true: ADP reduces the affinity of DnaK for GrpE. The energetic contributions of GrpE side-chains to GrpE-DnaK binding were probed by alanine-scanning mutagenesis. Sedimentation velocity (SV) analytical ultracentrifugation (AUC) was used to measure the equilibrium constants (Keq) for GrpE binding to the ATPase domain of DnaK in the presence of ADP. ADP-bound DnaK is the natural target of GrpE, and the addition of ADP (final concentration of 5 microM) to the preformed GrpE-DnaK(ATPase) complexes allowed the equilibrium association constants to be brought into an experimentally accessible range. Under these experimental conditions, the substitution of one single GrpE amino acid residue, arginine 183 with alanine, resulted in a GrpE-DnaK(ATPase) complex that was weakly associated (Keq =9.4 x 10(4) M). This residue has been previously shown to be part of a thermodynamic linkage between two structural domains of GrpE: the thermosensing long helices and the C-terminal beta-domains. Several other GrpE side-chains were found to have a significant change in the free energy of binding (DeltaDeltaG approximately 1.5 to 1.7 kcal mol(-1)), compared to wild-type GrpE.DnaK(ATPase) in the same experimental conditions. Overall, the strong interactions between GrpE and DnaK appear to be dominated by electrostatics, not unlike barnase and barstar, another well-characterized protein-protein interaction. GrpE, an inherent thermosensor, exhibits non-Arrhenius behavior with respect to its nucleotide exchange function at bacterial heat shock temperatures, and mutation of several solvent-exposed side-chains located along the thermosensing indicated that these residues are indeed important for GrpE-DnaK interactions.

Thermodynamic linkage in the GrpE nucleotide exchange factor, a molecular thermosensor.

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.

A structure-based interpretation of E.coli GrpE thermodynamic properties.

GrpE is the nucleotide exchange factor for the Escherichia coli molecular chaperone DnaK, the prokaryotic homologue of Hsp70. Thermodynamic properties of GrpE structural domains were characterized by examining a number of structural and point mutants using circular dichroism, differential scanning calorimetry and analytical ultracentrifugation. These structural domains are the long paired N-terminal helices, the central four-helix bundle, and the C-terminal compact beta-domains. We show that the central four-helix bundle (t(m) approximately 75 degrees C) provides a stable platform for the association of the long paired N-terminal helices (t(m) approximately 50 degrees C), which can then function as a temperature sensor. The stability of the N-terminal helices is linked to the presence of the C-terminal compact beta-domains of GrpE, providing a potential mechanism for coupling of DnaK-binding activity of GrpE with temperature. On the basis of our thermodynamic analysis of E.coli GrpE, we present a structure-based model for the melting properties of the nucleotide exchange factor, wherein the long paired helices function as a molecular thermocouple.

Ephs and ephrins close ranks.

The past decade has seen remarkable advances in identification of the proteins regulating axon guidance and synapse formation. Understanding the structural and molecular basis of their signaling properties is now the task at hand. The recently characterized crystal structure of the complex formed between the ligand-binding domain of EphB2 and the ectodomain of its binding partner ephrin-B2 provides an insight into the recognition and signal transduction mechanisms of this large multifunctional family of surface receptors. This heterotetrameric complex reveals a cyclic arrangement of subunits not previously seen in any receptor-ligand structure, and provides a basis for class specificity of binding.