Molecular dynamics of HIV-1 reverse transcriptase indicates increased flexibility upon DNA binding.

HIV-1 reverse transcriptase (RT) is one of the main targets for drugs used in the treatment of AIDS, among them, the non-nucleoside RT inhibitors (NNRTIs). The flexibility of RT unliganded and complexed to double-stranded DNA (RT/dsDNA), in water, has been studied by means of molecular dynamics. The simulations show that RT flexibility depends on its ligation state. The RT/dsDNA trajectories show larger fluctuations in the atomic positions than uncomplexed RT, particularly at the tips of the p66 fingers and thumb subdomains. This increased flexibility is consistent with the ability of the p66 fingers of the RT/dsDNA complex to close down after the binding of a deoxynucleoside triphosphate (dNTP) molecule, as observed in the crystal structures of RT/dsDNA bound to dNTP. The two complexation states present different patterns of concerted motions, indicating that the bound dsDNA alters RT flexibility. The motions of amino acid residues that form the non-nucleoside RT inhibitor binding pocket upon complexation with a NNRTI are anticorrelated with the p66 fingers (in RT/dsDNA) and correlated to the RNase H subdomain (unliganded RT). These concerted motions indicate that binding of a NNRTI could alter the flexibility of the subdomains whose motions are correlated to those of the binding pocket.

NMR reveals hydrogen bonds between oxygen and distal histidines in oxyhemoglobin.

Compared with free heme, the proteins hemoglobin (Hb) and myoglobin (Mb) exhibit greatly enhanced affinity for oxygen relative to carbon monoxide. This physiologically vital property has been attributed to either steric hindrance of CO or stabilization of O(2) binding by a hydrogen bond with the distal histidine. We report here the first direct evidence of such a hydrogen bond in both alpha- and beta-chains of oxyhemoglobin, as revealed by heteronuclear NMR spectra of chain-selectively labeled samples. Using these spectra, we have assigned the imidazole ring (1)H and (15)N chemical shifts of the proximal and distal histidines in both carbonmonoxy- and oxy-Hb. Because of their proximity to the heme, these chemical shifts are extremely sensitive to the heme pocket conformation. Comparison of the measured chemical shifts with values predicted from x-ray structures suggests differences between the solution and crystal structures of oxy-Hb. The chemical shift discrepancies could be accounted for by very small displacements of the proximal and distal histidines. This suggests that NMR could be used to obtain very high-resolution heme pocket structures of Hb, Mb, and other heme proteins.

Chain-selective isotopic labeling for NMR studies of large multimeric proteins: application to hemoglobin.

Multidimensional, multinuclear NMR has the potential to elucidate the mechanisms of allostery and cooperativity in multimeric proteins under near-physiological conditions. However, NMR studies of proteins made up of non-equivalent subunits face the problem of severe resonance overlap, which can prevent the unambiguous assignment of resonances, a necessary step in interpreting the spectra. We report the application of a chain-selective labeling technique, in which one type of subunit is labeled at a time, to carbonmonoxy-hemoglobin A (HbCO A). This labeling method can be used to extend previous resonance assignments of key amino acid residues, which are important to the physiological function of hemoglobin. Among these amino acid residues are the surface histidyls, which account for the majority of the Bohr effect. In the present work, we report the results of two-dimensional heteronuclear multiple quantum coherence (HMQC) experiments performed on recombinant (15)N-labeled HbCO A. In addition to the C2-proton (H epsilon(1)) chemical shifts, these spectra also reveal the corresponding C4-proton (H delta(2)) resonances, correlated with the N epsilon(2) and N delta(1) chemical shifts of all 13 surface histidines per alpha beta dimer. The HMQC spectrum also allows the assignment of the H delta(1), H epsilon(1), and N epsilon(1) resonances of all three tryptophan residues per alpha beta dimer in HbCO A. These results indicate that heteronuclear NMR, used with chain-selective isotopic labeling, can provide resonance assignments of key regions in large, multimeric proteins, suggesting an approach to elucidating the solution structure of hemoglobin, a protein with molecular weight 64.5 kDa.

Automated probabilistic method for assigning backbone resonances of (13C,15N)-labeled proteins.

We present a computer algorithm for the automated assignment of polypeptide backbone and 13C beta resonances of a protein of known primary sequence. Input to the algorithm consists of cross peaks from several 3D NMR experiments: HNCA, HN(CA)CO, HN(CA)HA, HNCACB, COCAH, HCA(CO)N, HNCO, HN(CO)CA, HN(COCA)HA, and CBCA(CO)NH. Data from these experiments performed on glutamine-binding protein are analyzed statistically using Bayes' theorem to yield objective probability scoring functions for matching chemical shifts. Such scoring is used in the first state of the algorithm to combine cross peaks from the first five experiments to form intraresidue segments of chemical shifts (Ni,HiN,Ci alpha, Ci beta, Ci'), while the latter five are combined into interresidue segments (Ci alpha,Ci beta,Ci',Ni + 1,Hi + 1N). Given a tentative assignment of segments, the second stage of the procedure calculates probability scores based on the likelihood of matching the chemical shifts of each segment with (i) overlapping segments; and (ii) chemical shift distributions of the underlying amino acid type (and secondary structure, if known). This joint probability is maximized by rearranging segments using a simulated annealing program, optimized for efficiency. The automated assignment program was tested using CBCANH and CBCA(CO)NH cross peaks of the two previously assigned proteins, calmodulin and CheA. The agreement between the results of our method and the published assignments was excellent. Our algorithm was also applied to the observed cross peaks of glutamine-binding protein of Escherichia coli, yielding an assignment in excellent agreement with that obtained by time-consuming, manual methods. The chemical shift assignment procedure described here should be most useful for NMR studies of large proteins, which are now feasible with the use of pulsed-field gradients and random partial deuteration of samples.

1H, 13C, and 15N NMR backbone assignments and chemical-shift-derived secondary structure of glutamine-binding protein of Escherichia coli.

1H, 13C, and 15N NMR assignments of the backbone atoms and beta-carbons have been made for liganded glutamine-binding protein (GlnBP) of Escherichia coli, a monomeric protein with 226 amino acid residues and a molecular weight of 24935 Da. GlnBP is a periplasmic binding protein which plays an essential role in the active transport of L-glutamine through the cytoplasmic membrane. The assignments have been obtained from three-dimensional triple-resonance NMR experiments on a 13C, 15N uniformly labeled sample as well as specifically labeled samples. Results from the 3D triple-resonance experiments, HNCO, HN(CO)CA, HN(COCA)HA, HNCA, HN(CA)HA, HN(CA)CO, and CBCA(CO)NH, are the main sources used to make the resonance assignments. Other 3D experiments, such as HNCACB, COCAH, HCACO, HCACON, and HOHAHA-HMQC, have been used to confirm the resonance assignments and to extend connections where resonance peaks are missing in some of the experiments mentioned above. We have assigned more than 95% of the polypeptide backbone resonances of GlnBP. The result of the standard manual assignment is in agreement with that predicted by an automated probailistic method developed in our laboratory. A solution secondary structure of the GlnBP-Gln complex has been proposed based on chemical shift deviations from random coil values. Eight alpha-helices and 10 beta-strands are derived using the Chemical Shift Index method.