Well-defined, stereospecific states in protein complexes are often in exchange with an ensemble of more dynamic orientations: the encounter states. The structure of the stereospecific complex between cytochrome P450cam and putidaredoxin was solved recently by X-ray diffraction as well as paramagnetic NMR spectroscopy. Other than the stereospecific complex, the NMR data clearly show the presence of additional states in the complex in solution. In these encounter states, populated for a small percentage of the time, putidaredoxin assumes multiple orientations and samples a large part of the surface of cytochrome P450cam. To characterize the nature of the encounter states, an extensive paramagnetic NMR dataset has been analyzed using the Maximum Occurrence of Regions methodology. The analysis reveals the location and maximal spatial extent of the additional states needed to fully explain the NMR data. Under the assumption of sparsity of the size of the conformational ensemble, several minor states can be located quite precisely. The distribution of these minor states correlates with the electrostatic potential map around cytochrome P450cam. Whereas some minor states are on isolated positively charged patches, others are connected to the stereospecific site via positively charged paths. The existence of electrostatically favorable pathways between the stereospecific interaction site and the different minor states or lack thereof suggests a means to discriminate between productive and futile encounter states.paramagnetic NMR | encounter complex | cytochrome P450cam | putidaredoxin | maximum occurrence C rystal structures suggest that proteins assume unique, stereospecific orientations within protein-protein complexes. However, a number of studies in solution have made clear that encounter states are an inherent element of protein complexes (1-8), especially in electron transfer (ET), where the interactions are often extremely fast (9). In the encounter complex, the proteins assume multiple other orientations, often in equilibrium with the major stereospecific state. In low-affinity complexes with dissociation constant (K d ) values > 10 μM, the encounter complex can represent a sizeable fraction, and in some cases, a well-defined, stereospecific complex may even be absent (10-15). The presence of encounter states may be a consequence of the chemical nature of proteins. In nonobligate stereospecific complexes, the interface represents a small fraction of the total protein surface, and therefore, it is reasonable to assume that weak interactions also occur elsewhere. In the case in which protein pairs have evolved to exhibit a high association rate by using electrostatic preorientation, electrostatic patches seem to enhance the presence of encounter states (16,17). On the one hand, the preorientation reduces the surface area that is visited by the partner, thus enhancing the number of productive encounters, but on the other hand, highly charged patches can bind the oppositely charged protein in many orientations with a...

Long-range NMR data, namely residual dipolar couplings (RDCs) from external alignment and paramagnetic data, are becoming increasingly popular for the characterization of conformational heterogeneity of multidomain biomacromolecules and protein complexes. The question addressed here is how much information is contained in these averaged data. We have analyzed and compared the information content of conformationally averaged RDCs caused by steric alignment and of both RDCs and pseudocontact shifts caused by paramagnetic alignment, and found that, despite the substantial differences, they contain a similar amount of information. Furthermore, using several synthetic tests we find that both sets of data are equally good towards recovering the major state(s) in conformational distributions.

The presence of heterogeneity in the interdomain arrangement of several biomolecules is required for their function. Here we present a method to obtain crucial clues to distinguish between different kinds of protein conformational distributions based on experimental NMR data. The method explores subregions of the conformational space and provides both upper and lower bounds of probability for the system to be in each subregion.

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DEER data improve the understanding of protein conformational landscapes.

Detecting conformational heterogeneity in biological macromolecules is a key for the understanding of their biological function. We here provide a comparison between two independent approaches to assess conformational heterogeneity: molecular dynamics simulations, performed without inclusion of any experimental data, and maximum occurrence (MaxOcc) distribution over the topologically available conformational space. The latter only reflects the extent of the averaging and identifies regions which are most compliant with the experimentally measured NMR Residual Dipolar Couplings (RDCs). The analysis was performed for the HIV-1 TAR RNA, consisting of two helical domains connected by a flexible bulge junction, for which four sets of RDCs were available as well as an 8.2 μs all-atom molecular dynamics simulation. A sample and select approach was previously applied to extract from the molecular dynamics trajectory conformational ensembles in agreement with the four sets of RDCs. The MaxOcc analysis performed here identifies the most likely sampled region in the conformational space of the system which, strikingly, overlaps well with the structures independently sampled in the molecular dynamics calculations and even better with the RDC selected ensemble.

Triazoloacridinone C-1305, a potent antitumor agent recommended for Phase I clinical trials, exhibits high activity towards a wide range of experimental colon carcinomas, in many cases associated with complete tumor regression. C-1305 is a well-established dsDNA intercalator, yet no information on its mode of binding into DNA is available to date. Herein, we present the NMR-driven and MD-refined reconstruction of the 3D structures of the d(CGA TAT CG) 2 :C-1305 and d(CCC TAG GG) 2 :C-1305 non-covalent adducts. In both cases, the ligand intercalates at the TA/TA site, forming well-defined dsDNA:drug 1:1 mol/mol complexes. Orientation of the ligand within the binding site was unambiguously established by the DNA/ligand proton-proton NOE contacts. A subsequent, NMR-driven study of the sequence-specificity of C-1305 using a series of DNA duplexes, allowed us to confirm a strong preference towards TA/TA dinucleotide steps, followed by the TG/CA steps. Interestingly, no interaction at all was observed with duplexes containing exclusively the AT/AT, GG/ cc and GA/tc steps. DNA-directed, rational antineoplastic agent development has made an extensive use of the acridine pharmacophore 1. Among many diverse families of acridine derivatives, development of a series of novel triazoloacridinones with potent antitumor activities has been started some time ago 2. The most active triazoloacridinone derivative obtained to date, 5-[[3-(dimethylamino)propyl]amino]-8-hydroxy-6H-v-triazolo[4,5,1-de] acridin-6-one, codenamed C-1305 (Fig. 1), showed high antitumor activity towards a wide range of different experimental tumors in vitro and in vivo, including both murine and human colon carcinomas, which in most cases was associated with complete tumor regression 3. Induction of apoptosis in human leukemia cells after uptake of C-1305 has also been demonstrated 4,5. Its interaction with several molecular targets 6,7 , as well as its metabolism 8-10 , were extensively studied. For instance, this compound was shown to be a topoisomerase II poison, which stabilizes unusually toxic covalent complexes between DNA and the enzyme 11. It was also demonstrated that C-1305 is a viable double stranded DNA (dsDNA) intercalator, yet no sequence-specificity of this potential drug was revealed since UV, CD and ELD studies have shown that it intercalated into ctDNA, p(dAdT) 2 and p(dGdC) 2 polymers at the same ratio 12. Moreover, chemical probing with DEPC, combined with molecular modelling studies suggested that C-1305 is able to induce substantial distortions of a dsDNA duplex while intercalating into the GGG triplets, which is a unique effect among the known topoisomerase II inhibitors.

Uridine tetrads (U-tetrads) are a structural element encountered in RNA G-quadruplexes, for example, in the structures formed by the biologically relevant human telomeric repeat RNA. For these molecules, an unexpectedly strong stabilizing influence of a U-tetrad forming at the 3' terminus of a quadruplex was reported. Here we present the high resolution solution NMR structure of the r(UGGUGGU) 4 quadruplex which, in our opinion, provides an explanation for this stabilization. Our structure features a distinctive, abrupt chain reversal just prior to the 3' uridine tetrad. Similar 'reversed U-tetrads' were already observed in the