The E2 proteins are transcription/replication factors from papillomaviruses. Human papillomaviruses (HPVs) can be broadly divided in two groups; low-risk HPV subtypes cause benign warts while high-risk HPVs give rise to cervical cancer. Although a range of crystal structures of E2 DNA-binding domains (DBD) from both high- and low-risk HPV subtypes have been reported previously, structures of E2 DBD:DNA complexes have only been available for high-risk HPV18 and bovine papillomavirus (BPV1). In the present study we report the unliganded and DNA complex structures of the E2 DBD from the low-risk HPV6. As in the previous E2–DNA structures, complex formation results in considerable bending of the DNA, which is facilitated by sequences with A:T-rich spacers that adopt a pre-bent conformation. The low-risk HPV6 E2–DNA complex differs from the earlier structures in that minimal deformation of the protein accompanies complex formation. Stopped-flow kinetic studies confirm that both high- and low-risk E2 proteins adapt their structures on binding to DNA, although this is achieved more readily for HPV6 E2. It therefore appears that the higher selectivity of the HPV6 E2 protein may arise from its limited molecular adaptability, a property that might distinguish the behaviour of E2 proteins from high- and low-risk HPV subtypes.

The spectral properties of emissive photoproducts, formed upon 633 nm irradiation of a terrylene diimide dye, were investigated. Ensemble and single-molecule level experiments were conducted by immobilizing the TDI dye molecules in a polystyrene film. In the bulk experiments, green emission could be observed from the photobleached areas (photobleached with 633 nm light) when excited with 480 or 514 nm light. Similar phenomena were also observed at the single-molecule level. On the basis of the single-molecule experiments, a conversion efficiency of about 5% was estimated for the formation of emissive spectrally blue-shifted photoproducts. These green emissive photoproducts have spectral properties that resemble those of lower rylene homologues, e.g. perylene diimide or perylene monoimide. Our results indicate that the formation of blue-shifted emissive photoproducts can have implications for analyzing single-molecule FRET experiments or multiple color-labeled fluorescent systems.

To cite this article: Hooley E, McEwan PA, Emsley J. Molecular modeling of the prekallikrein structure provides insights into high-molecular-weight kininogen binding and zymogen activation. J Thromb Haemost 2007; 5: 2461-6. Summary. Background: Prekallikrein (PK) plays a central role in the contact system that activates blood coagulation and is involved in the regulation of blood pressure. Objectives: To provide three-dimensional structural data for PK and rationalize the molecular basis of substrate recognition and zymogen activation. Patients/methods: The PK homology model was constructed using the coagulation factor (F) XI crystal structure as a template with the program SWISS-MODEL. Results: The domain organization of the PK apple domains and serine protease is conserved compared to FXI. Surface charge calculations on the PK model revealed that ligand binding to high-molecular-weight kininogen (HK) is predicted to have two key determinants: a pocket within the apple 2 domain and a basic channel formed at the interface of apple domains 1 and 4. A hereditary mutation resulting in PK deficiency (Gly104Arg) and the Lys140 a-kallikrein cleavage site both disrupt HK binding and are shown to map to opposite sides of the apple 2 domain pocket. The model also describes the differences in the apple 4 domain that prevents dimer formation in PK vs. FXI. A C-terminal extension in the PK serine protease domain is described as a potential substrate for prolylcarboxypeptidase. Conclusions: The interaction between PK and HK is mediated by two discrete surfaces formed by the PK A1, A2 and A4 domains with charge likely to be a critical component of the binding. A novel mode of PK activation is postulated to involve prolylcarboxypeptidase cleaving at the C-terminus rather than the activation loop.

DNA-stabilized silver nanoclusters (DNA-AgNCs) are promising fluorophores whose photophysical properties and synthesis procedures have received increased attention in the literature. However, depending on the preparation conditions and the DNA sequence, the DNA-AgNC samples can host a range of different emitters, which can influence the reproducibility of the optical response and the evolution over time of the populations of these emitters. We have developed a simple method to characterize the spectral heterogeneity and time evolution of these emissive species at any given point in time after preparation, by plotting the average decay time as a function of emission wavelength. These so-called average decay time spectra were acquired for different excitation wavelengths of AgNCs stabilized by an oligonucleotide containing 24 cytosines (C24-AgNCs). The average decay time spectra allowed the comparison of sample preparation and the judgment of reproducibility. Therefore, we propose the use of the average decay time spectra as a robust and easy tool to characterize and compare different as-synthesized DNA-AgNC samples. The average decay time spectra can in general also be used to characterize the spectral heterogeneity of other fluorophores, such as luminescent colloidal nanoparticles, and to assess the reproducibility of a synthetic procedure containing an unknown distribution of emissive species.

Aggretin is a C-type lectin purified from Calloselasma rhodostoma snake venom. It is a potent activator of platelets, resulting in a collagen-like response by binding and clustering platelet receptor CLEC-2. We present here the crystal structure of aggretin at 1.7 A which reveals a unique tetrameric quaternary structure. The two alphabeta heterodimers are arranged through 2-fold rotational symmetry, resulting in an antiparallel side-by-side arrangement. Aggretin thus presents two ligand binding sites on one surface and can therefore cluster ligands in a manner reminiscent of convulxin and flavocetin. To examine the molecular basis of the interaction with CLEC-2, we used a molecular modeling approach of docking the aggretin alphabeta structure with the CLEC-2 N-terminal domain (CLEC-2N). This model positions the CLEC-2N structure face down in the "saddle"-shaped binding site which lies between the aggretin alpha and beta lectin-like domains. A 2-fold rotation of this complex to generate the aggretin tetramer reveals dimer contacts for CLEC-2N which bring the N- and C-termini into the proximity of each other, and a series of contacts involving two interlocking beta-strands close to the N-terminus are described. A comparison with homologous lectin-like domains from the immunoreceptor family reveals a similar but not identical dimerization mode, suggesting this structure may represent the clustered form of CLEC-2 capable of signaling across the platelet membrane.