We present a working model of the flap-opening mechanism in free HIV-1 protease which involves a transition from a semi-open to an open conformation that is facilitated by interaction of the Phe53 ring with the substrate. We also identify a surprising fluctuation of the beta-sheet intermonomer interface that suggests a structural requirement for maturation of the protease. Thus, slow conformational fluctuations identified by (1)H and (15)N transverse relaxation measurements can be related to the biological functions of proteins.
Crystal structures have shown that the HIV-1 protease flaps, domains that control access to the active site, are closed when the active site is occupied by a ligand. Although flap structures ranging from closed to semi-open are observed in the free protease, crystal structures reveal that even the semi-open flaps block access to the active site, indicating that the flaps are mobile in solution. The goals of this paper are to characterize the secondary structure and fast (sub-ns) dynamics of the flaps of the free protease in solution, to relate these results to X-ray structures and to compare them with predictions of dynamics calculations. To this end we have obtained nearly complete backbone and many sidechain signal assignments of a fully active free-protease construct that is stabilized against autoproteolysis by three point mutations. The secondary structure of this protein was characterized using the chemical shift index, measurements of 3h J NCЈ couplings across hydrogen bonds, and NOESY connectivities. Analysis of these measurements indicates that the protease secondary structure becomes irregular near the flap tips, residues 49-53. Model-free analysis of 15 N relaxation parameters, T 1 , T 2 (T 1 ) and 15 N-{ 1 H} NOE, shows that residues in the flap tips are flexible on the sub-ns time scale, in contrast with previous observations on the inhibitor-bound protease. These results are compared with theoretical predictions of flap dynamics and the possible biological significance of the sub-ns time scale dynamics of the flap tips is discussed.
SUMMARY Nuclear export of unspliced and singly spliced viral mRNA is a critical step in the HIV life cycle. The structural basis by which the virus selects its own mRNA among more abundant host cellular RNAs for export has been a mystery for more than 25 years. Here, we describe an unusual topological structure that the virus uses to recognize its own mRNA. The viral Rev response element (RRE) adopts an “A”-like structure in which the two legs constitute two tracks of binding sites for the viral Rev protein and position the two primary known Rev-binding sites ~55 Å apart, matching the distance between the two RNA-binding motifs in the Rev dimer. Both the legs of the “A” and the separation between them are required for optimal RRE function. This structure accounts for the specificity of Rev for the RRE and thus the specific recognition of the viral RNA.
Copper is an essential transition metal for living organisms but it is detrimental in excess. The metalloregulatory protein copper‐sensing operon repressor (CsoR) in bacteria has evolved to prevent cytoplasmic copper toxicity. Cu(I)‐binding to tetrameric CsoRs mediates transcriptional derepression of copper resistance genes but the mechanism is unknown. A phylogenetic analysis of 227 DUF156 protein members including biochemically or structurally characterized CsoR/RcnR repressors reveals that Geobacillus thermodenitrificans (Gt) CsoR characterized here is representative of CsoRs from pathogenic bacilli Listeria monocytogenes and Bacillus anthracis. The 2.56 Å structure of Cu(I)‐bound Gt CsoR reveals that Cu(I) binding induces a kink in the α2‐helix between two conserved copper‐ligating residues and folds an N‐terminal tail (residues 12‐19) over the Cu(I) binding site. NMR studies of Gt CsoR reveal that this tail is flexible in the apo‐state with these dynamics quenched upon Cu(I) binding. Small angle X‐ray scattering (SAXS) experiments on an N‐terminally truncated Gt CsoR (∆2‐10) reveal that the Cu(I)‐bound tetramer is hydrodynamically more compact than is the apo‐state. A mutational analysis of residues critical to N‐terminal tail folding reveals that these residues function to stabilize the apoprotein‐DNA complex and/or control the extent of allosteric negative regulation of DNA binding by Cu(I), but to varying degrees. The mechanism of Cu(I)‐mediated allosteric switching in CsoRs is discussed. Grant Funding Source: Supported by NIH grant GM042569
In order to improve the design of HIV-1 protease inhibitors, it is essential to understand how they interact with active site residues, particularly the catalytic Asp25 and Asp125 residues. KNI-272 is a promising, potent HIV-1 protease inhibitor (K(i) approximately 5 pM), currently undergoing phase 1 clinical trials. Because KNI-272 is asymmetric, the complex it forms with the homodimeric HIV-1 protease also lacks symmetry, and the two protease monomers can have distinct NMR spectra. Monomer specific signal assignments were obtained for amino acid residues in the drug binding site as well as for six of the eight Asp residues in the protease/KNI-272 complex. Using these assignments, the ionization states of the Asp carboxyl groups were determined from measurements of (a) the pD dependence of the chemical shifts of the Asp carboxyl carbons and (b) the H/D isotope effect upon the Asp carboxyl carbon chemical shifts. The results of these measurements indicate that the carboxyl of Asp25 is protonated while that of Asp125 is not protonated. These findings provide not only the first experimental evidence regarding the distinct protonation states of Asp25/125 in HIV-1 protease/drug complexes, but also shed light on interactions responsible for inhibitor binding that should form the basis for improved drug designs.
Na+/Ca2+ exchanger (NCX) proteins mediate Ca2+-fluxes across the cell membrane to maintain Ca2+ homeostasis in many cell types. Eukaryotic NCX contains Ca2+-binding regulatory domains, CBD1 and CBD2. Ca2+ binding to a primary sensor (Ca3-Ca4 sites) on CBD1 activates mammalian NCXs, whereas CALX, a Drosophila NCX ortholog, displays an inhibitory response to regulatory Ca2+. To further elucidate the underlying regulatory mechanisms, we determined the 2.7 Å crystal structure of mammalian CBD12-E454K, a two-domain construct that retains wild-type properties. In conjunction with stopped-flow kinetics and SAXS (small-angle X-ray scattering) analyses of CBD12 mutants, we show that Ca2+ binding to Ca3-Ca4 sites tethers the domains via a network of interdomain salt-bridges. This Ca2+-driven interdomain switch controls slow dissociation of “occluded” Ca2+ from the primary sensor and thus dictates Ca2+ sensing dynamics. In the Ca2+-bound conformation, the interdomain angle of CBD12 is very similar in NCX and CALX, meaning that the interdomain distances cannot account for regulatory diversity in NCX and CALX. Since the two-domain interface is nearly identical among eukaryotic NCXs, including CALX, we suggest that the Ca2+-driven interdomain switch described here represents a general mechanism for initial conduction of regulatory signals in NCX variants.
The 3 0 untranslated region (3 0 UTR) of turnip crinkle virus (TCV) genomic RNA contains a cap-independent translation element (CITE), which includes a ribosome-binding structural element (RBSE) that participates in recruitment of the large ribosomal subunit. In addition, a large symmetric loop in the RBSE plays a key role in coordinating the incompatible processes of viral translation and replication, which require enzyme progression in opposite directions on the viral template. To understand the structural basis for the large ribosomal subunit recruitment and the intricate interplay among different parts of the molecule, we determined the global structure of the 102-nt RBSE RNA using solution NMR and small-angle x-ray scattering. This RNA has many structural features that resemble those of a tRNA in solution. The hairpins H1 and H2, linked by a 7-nucleotide linker, form the upper part of RBSE and hairpin H3 is relatively independent from the rest of the structure and is accessible to interactions. This global structure provides insights into the three-dimensional layout for ribosome binding, which may serve as a structural basis for its involvement in recruitment of the large ribosomal subunit and the switch between viral translation and replication. The experimentally determined threedimensional structure of a functional element in the 3 0 UTR of an RNA from any organism has not been previously reported. The RBSE structure represents a prototype structure of a new class of RNA structural elements involved in viral translation/replication processes.new method | NMR | SAXS | 3' UTR RNA | RNA structure S tructural elements in mRNAs such as the 5 0 cap, internal ribosome entry site in the 5 0 UTR, and the 3 0 poly(A) tail in the 3 0 UTR are important determinants for efficient translation initiation (1). These structural elements can function synergistically to attract ribosomes and translation factors to enhance translation initiation (2, 3). In both cap and poly(A) tail-dependent translation in eukaryotes this enhancement is realized by the binding factor elF4G associating with both the polyA binding protein (Pab1p) and elF4E, resulting in a circularized mRNA template (4), which has been visualized under atomic force microscopy (5). In contrast, high level initiation of capindependent translation in many plant viruses involves 3 0 UTR RNA elements known as CITE (6). TCV lacks a 5 0 cap and poly(A) tail. Instead the virus uses a structural element in its 3 0 UTR that synergistically enhances translation when associated with its 5 0 UTR (7) (Fig. 1A). A model for 3 0 UTR involvement in the recruitment of the large ribosomal subunit has been proposed to account for the enhancement (7) but the experimental structural basis for such an involvement has not been demonstrated. Moreover, mechanisms are required to temporally coordinate viral replication and translation because these processes are mutually exclusive due to the opposing directions of protein and RNA synthesis. The infecting genomic RNA must first be trans...
We report a "top-down" method that uses mainly duplexes' global orientations and overall molecular dimension and shape restraints, which were extracted from experimental NMR and small angle Xray scattering (SAXS) data respectively, to determine global architectures of RNA molecules consisting of mostly A-form like duplexes. The method is implemented in the G2G (from Global measurement to Global Structure) toolkit of programs. We demonstrate the efficiency and accuracy of the method by determining the global structure of a 71-nucleotide RNA using experimental data. The backbone root-mean-square-deviation (RMSD) of the ensemble of the calculated global structures relative to the X-ray crystal structure using the experimental data is 3.0 ± 0.3 Å, and the RMSD is only 2.5 ± 0.2 Å for the three duplexes that were orientation-restrained during the calculation. The global structure simplifies interpretation of multi-dimensional nuclear Overhauser spectra for high resolution structure determination. The potential general application of the method for RNA structure determination is discussed.To whom correspondence should be addressed: Y-X Wang: wangyu@ncifcrf.gov, (phone) 301-846-5985. * These authors contributed equally to this work & Current address: Gynecology and Obstetric Hospital, Fudan University, 419 Fangxie Road, Shanghai 200011, People's Republic of China Supporting Information Available: A more detailed description of the methods and materials, including a detailed description of the G2G toolkit is provided in the Supporting Information. The calculation protocols, and the G2G toolkit package, all data files together with the coordinates and restraint filescan also be downloaded from the authors' web sites http://xxx.
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