Identification of drivers for the metamorphic transition of HIV-1 reverse transcriptase.

Recent structural characterizations of the p51 and p66 monomers have established an important starting point for understanding the maturation pathway of the human immunodeficiency virus (HIV)-1 reverse transcriptase p66/p51 heterodimer. This process requires a metamorphic transition of the polymerase domain leading to formation of a p66/p66' homodimer that exists as a structural heterodimer. To better understand the drivers for this metamorphic transition, we have performed NMR studies of 15N-labeled RT216 - a construct that includes the fingers and most of the palm domains. These studies are consistent with the conclusion that the p66 monomer exists as a spring-loaded complex. Initial dissociation of the fingers/palm : connection complex allows the fingers/palm to adopt an alternate, more stable structure, reducing the rate of reassociation and facilitating subsequent maturation steps. One of the drivers for an initial extension of the fingers/palm domains is identified as a straightening of helix E relative to its conformation in the monomer by eliminating a bend of ∼50° near residue Phe160. NMR and circular dichroism data also are consistent with the conclusion that a hydrophobic surface of palm domain that becomes exposed after the initial dissociation, as well as the intrinsic conformational preferences of the palm domain C-terminal segment, facilitates the formation of the ß-sheet structure that is unique to the active polymerase subunit. Spectral comparisons based on 15N-labeled constructs are all consistent with previous structural conclusions based on studies of 13C-methyl-labeled constructs.

Unfolding the HIV-1 reverse transcriptase RNase H domain--how to lose a molecular tug-of-war.

Formation of the mature HIV-1 reverse transcriptase (RT) p66/p51 heterodimer requires subunit-specific processing of the p66/p66' homodimer precursor. Since the ribonuclease H (RH) domain contains an occult cleavage site located near its center, cleavage must occur either prior to folding or subsequent to unfolding. Recent NMR studies have identified a slow, subunit-specific RH domain unfolding process proposed to result from a residue tug-of-war between the polymerase and RH domains on the functionally inactive, p66' subunit. Here, we describe a structural comparison of the isolated RH domain with a domain swapped RH dimer that reveals several intrinsically destabilizing characteristics of the isolated domain that facilitate excursions of Tyr427 from its binding pocket and separation of helices B and D. These studies provide independent support for the subunit-selective RH domain unfolding pathway in which instability of the Tyr427 binding pocket facilitates its release followed by domain transfer, acting as a trigger for further RH domain destabilization and subsequent unfolding. As further support for this pathway, NMR studies demonstrate that addition of an RH active site-directed isoquinolone ligand retards the subunit-selective RH' domain unfolding behavior of the p66/p66' homodimer. This study demonstrates the feasibility of directly targeting RT maturation with therapeutics.

Asymmetric conformational maturation of HIV-1 reverse transcriptase.

HIV-1 reverse transcriptase utilizes a metamorphic polymerase domain that is able to adopt two alternate structures that fulfill catalytic and structural roles, thereby minimizing its coding requirements. This ambiguity introduces folding challenges that are met by a complex maturation process. We have investigated this conformational maturation using NMR studies of methyl-labeled RT for the slower processes in combination with molecular dynamics simulations for rapid processes. Starting from an inactive conformation, the p66 precursor undergoes a unimolecular isomerization to a structure similar to its active form, exposing a large hydrophobic surface that facilitates initial homodimer formation. The resulting p66/p66' homodimer exists as a conformational heterodimer, after which a series of conformational adjustments on different time scales can be observed. Formation of the inter-subunit RH:thumb' interface occurs at an early stage, while maturation of the connection' and unfolding of the RH' domains are linked and occur on a much slower time scale.

Selective unfolding of one Ribonuclease H domain of HIV reverse transcriptase is linked to homodimer formation.

HIV-1 reverse transcriptase (RT), a critical enzyme of the HIV life cycle and an important drug target, undergoes complex and largely uncharacterized conformational rearrangements that underlie its asymmetric folding, dimerization and subunit-selective ribonuclease H domain (RH) proteolysis. In the present article we have used a combination of NMR spectroscopy, small angle X-ray scattering and X-ray crystallography to characterize the p51 and p66 monomers and the conformational maturation of the p66/p66' homodimer. The p66 monomer exists as a loosely structured molecule in which the fingers/palm/connection, thumb and RH substructures are connected by flexible (disordered) linking segments. The initially observed homodimer is asymmetric and includes two fully folded RH domains, while exhibiting other conformational features similar to that of the RT heterodimer. The RH' domain of the p66' subunit undergoes selective unfolding with time constant ∼6.5 h, consistent with destabilization due to residue transfer to the polymerase' domain on the p66' subunit. A simultaneous increase in the intensity of resonances near the random coil positions is characterized by a similar time constant. Consistent with the residue transfer hypothesis, a construct of the isolated RH domain lacking the two N-terminal residues is shown to exhibit reduced stability. These results demonstrate that RH' unfolding is coupled to homodimer formation.

Protein-mediated antagonism between HIV reverse transcriptase ligands nevirapine and MgATP.

Nonnucleoside reverse transcriptase inhibitors (NNRTIs) play a central role in the treatment of AIDS, but their mechanisms of action are incompletely understood. The interaction of the NNRTI nevirapine (NVP) with HIV-1 reverse transcriptase (RT) is characterized by a preference for the open conformation of the fingers/thumb subdomains, and a reported variation of three orders of magnitude between the binding affinity of NVP for RT in the presence or absence of primer/template DNA. To investigate the relationship between conformation and ligand binding, we evaluated the use of methionine NMR probes positioned near the tip of the fingers or thumb subdomains. Such probes would be expected to be sensitive to changes in the local environment depending on the fractions of open and closed RT. Comparisons of the NMR spectra of three conservative mutations, I63M, L74M, and L289M, indicated that M63 showed the greatest shift sensitivity to the addition of NVP. The exchange kinetics of the M63 resonance are fast on the chemical shift timescale, but become slow in the presence of NVP due to the slow binding of RT with the inhibitor. The simplest model consistent with this behavior involves a rapid open/closed equilibrium coupled with a slow interaction of the inhibitor with the open conformation. Studies of RT in the presence of both NVP and MgATP indicate a strong negative cooperativity. Binding of MgATP reduces the fraction of RT bound to NVP, as indicated by the intensity of the NVP-perturbed M230 resonance, and enhances the dissociation rate constant of the NVP, resulting in an increase of the open/closed interconversion rate, so that the M63 resonance moves into the fast/intermediate-exchange regime. Protein-mediated interactions appear to explain most of the affinity variation of NVP for RT.

Crystal structure of calmodulin binding domain of orai1 in complex with Ca2+ calmodulin displays a unique binding mode.

Orai1 is a plasma membrane protein that in its tetrameric form is responsible for calcium influx from the extracellular environment into the cytosol in response to interaction with the Ca(2+)-depletion sensor STIM1. This is followed by a fast Ca(2+)·calmodulin (CaM)-dependent inhibition, resulting from CaM binding to an Orai1 region called the calmodulin binding domain (CMBD). The interaction between Orai1 and CaM at the atomic level remains unknown. Here, we report the crystal structure of a CaM·Orai1-CMBD complex showing one CMBD bound to the C-terminal lobe of CaM, differing from other CaM-target protein complexes, in which both N- and C-terminal lobes of CaM (CaM-N and CaM-C) are involved in target binding. Orai1-CMBD binds CaM-C mainly through hydrophobic interactions, primarily involving residue Trp(76) of Orai1-CMBD, which interacts with the hydrophobic pocket of CaM-C. However, NMR data, isothermal titration calorimetry data, and pulldown assays indicated that CaM-N and CaM-C both can bind Orai1-CMBD, with CaM-N having ∼4 times weaker affinity than CaM-C. Pulldown assays of a Orai1-CMBD(W76E) mutant, gel filtration chromatography data, and NOE signals indicated that CaM-N and CaM-C can each bind one Orai1-CMBD. Thus our studies support an unusual, extended 1:2 binding mode of CaM to Orai1-CMBDs, and quantify the affinity of Orai1 for CaM. We propose a two-step mechanism for CaM-dependent Orai1 inactivation initiated by binding of the C-lobe of CaM to the CMBD of one Orai1 followed by the binding of the N-lobe of CaM to the CMBD of a neighboring Orai1.

Metal and ligand binding to the HIV-RNase H active site are remotely monitored by Ile556.

HIV-1 reverse transcriptase (RT) contains a C-terminal ribonuclease H (RH) domain on its p66 subunit that can be expressed as a stable, although inactive protein. Recent studies of several RH enzymes demonstrate that substrate binding plays a major role in the creation of the active site. In the absence of substrate, the C-terminal helix E of the RT RNase H domain is dynamic, characterized by severe exchange broadening of its backbone amide resonances, so that the solution characterization of this region of the protein has been limited. Nuclear magnetic resonance studies of 13C-labeled RH as a function of experimental conditions reveal that the δ1 methyl resonance of Ile556, located in a short, random coil segment following helix E, experiences a large 13C shift corresponding to a conformational change of Ile556 that results from packing of helix E against the central ß-sheet. This shift provides a useful basis for monitoring the effects of various ligands on active site formation. Additionally, we report that the RNase H complexes formed with one or both divalent ions can be individually observed and characterized using diamagnetic Zn2+ as a substitute for Mg2+. Ordering of helix E results specifically from the interaction with the lower affinity binding to the A divalent ion site.

The NAD(+)-dependent protein deacetylase activity of SIRT1 is regulated by its oligomeric status.

SIRT1, a NAD(+)-dependent protein deacetylase, is an important regulator in cellular stress response and energy metabolism. While the list of SIRT1 substrates is growing, how the activity of SIRT1 is regulated remains unclear. We have previously reported that SIRT1 is activated by phosphorylation at a conserved Thr522 residue in response to environmental stress. Here we demonstrate that phosphorylation of Thr522 activates SIRT1 through modulation of its oligomeric status. We provide evidence that nonphosphorylated SIRT1 protein is aggregation-prone in vitro and in cultured cells. Conversely, phosphorylated SIRT1 protein is largely in the monomeric state and more active. Our findings reveal a novel mechanism for environmental regulation of SIRT1 activity, which may have important implications in understanding the molecular mechanism of stress response, cell survival, and aging.

Conformational dependence of 13C shielding and coupling constants for methionine methyl groups.

Methionine residues fulfill a broad range of roles in protein function related to conformational plasticity, ligand binding, and sensing/mediating the effects of oxidative stress. A high degree of internal mobility, intrinsic detection sensitivity of the methyl group, and low copy number have made methionine labeling a popular approach for NMR investigation of selectively labeled protein macromolecules. However, selective labeling approaches are subject to more limited information content. In order to optimize the information available from such studies, we have performed DFT calculations on model systems to evaluate the conformational dependence of (3)J (CSCC), (3)J (CSCH), and the isotropic shielding, sigma(iso). Results have been compared with experimental data reported in the literature, as well as data obtained on [methyl-(13)C]methionine and on model compounds. These studies indicate that relative to oxygen, the presence of the sulfur atom in the coupling pathway results in a significantly smaller coupling constant, (3)J (CSCC)/(3)J (COCC) approximately 0.7. It is further demonstrated that the (3)J (CSCH) coupling constant depends primarily on the subtended CSCH dihedral angle, and secondarily on the CSCC dihedral angle. Comparison of theoretical shielding calculations with the experimental shift range of the methyl group for methionine residues in proteins supports the conclusion that the intra-residue conformationally-dependent shift perturbation is the dominant determinant of delta(13)Cepsilon. Analysis of calmodulin data based on these calculations indicates that several residues adopt non-standard rotamers characterized by very large approximately 100 degrees chi(3) values. The utility of the delta(13)Cepsilon as a basis for estimating the gauche/trans ratio for chi(3) is evaluated, and physical and technical factors that limit the accuracy of both the NMR and crystallographic analyses are discussed.

Homodimerization of the p51 subunit of HIV-1 reverse transcriptase.

The dimerization of HIV reverse transcriptase (RT), required to obtain the active form of the enzyme, is influenced by mutations, non-nucleoside reverse transcriptase inhibitors (NNRTIs), nucleotide substrates, Mg ions, temperature, and specifically designed dimerization inhibitors. In this study, we have utilized nuclear magnetic resonance (NMR) spectroscopy of the [methyl-(13)C]methionine-labeled enzyme and small-angle X-ray scattering (SAXS) to investigate how several of these factors influence the dimerization behavior of the p51 subunit. The (1)H-(13)C HSQC spectrum of p51 obtained at micromolar concentrations indicates that a significant fraction of the p51 adopts a "p66-like" conformation. SAXS data obtained for p51 samples were used to determine the fractions of monomer and dimer in the sample and to evaluate the conformation of the fingers/thumb subdomain. All of the p51 monomer observed was found to adopt the compact, "p51C" conformation observed for the p51 subunit in the RT heterodimer. The NMR and SAXS data indicate that the p51 homodimer adopts a structure that is similar to the p66/p51 heterodimer, with one p51C subunit and a second p51 subunit in an extended, "p51E" conformation that resembles the p66 subunit of the heterodimer. The fractional dimer concentration and the fingers/thumb orientation are found to depend strongly on the experimental conditions and exhibit a qualitative dependence on nevirapine and ionic strength (KCl) that is similar to the behavior reported for the heterodimer and the p66 homodimer. The L289K mutation interferes with p51 homodimer formation as it does with formation of the heterodimer, despite its location far from the dimer interface. This effect is readily interpreted in terms of a conformational selection model, in which p51(L289K) has a much greater preference for the compact, p51C conformation. A reduced level of dimer formation then results from the reduced ratio of the p51E(L289K) to p51C(L289K) monomers.

Solution characterization of [methyl-(13)C]methionine HIV-1 reverse transcriptase by NMR spectroscopy.

HIV reverse transcriptase (RT) is a primary target for drug intervention in the treatment of AIDS. We report the first solution NMR studies of [methyl-(13)C]methionine HIV-1 RT, aimed at better understanding the conformational and dynamic characteristics of RT, both in the presence and absence of the non-nucleoside RT inhibitor (NNRTI) nevirapine. The selection of methionine as a structural probe was based both on its favorable NMR characteristics, and on the presence of two important active site methionine residues, M184(66) and M230(66). Observation of the M184 resonance is subunit dependent; in the p66 subunit the solvent-exposed residue produces a readily observed signal with a characteristic resonance shift, while in the globular p51 subunit, the M184(51) resonance is shifted and broadened as M184 becomes buried in the protein interior. In contrast, although structural data indicates that the environment of M230 is also strongly subunit dependent, the M230 resonances from both subunits have very similar shift and relaxation characteristics. A comparison of chemical shift and intensity data with model-based predictions gives reasonable agreement for M184(66), while M230(66), located on the beta-hairpin "primer grip", is more mobile and solvent-exposed than suggested by crystal structures of the apo enzyme which have a "closed" fingers-thumb conformation. This mobility of the primer grip is presumably important for binding of non-nucleoside RT inhibitors (NNRTIs), since the NNRTI binding pocket is not observed in the absence of the inhibitors, requiring instead that the binding pocket be dynamically accessible. In the presence of the nevirapine, both the M184(66) and M230(66) resonances are significantly perturbed, while none of the methionine resonances in the p51 subunit is sensitive to this inhibitor. Site-directed mutagenesis indicates that both M16 and M357 produce two resonances in each subunit, and for both residues, the intensity ratio of the component peaks is strongly subunit dependent. Conformational features that might explain the multiple peaks are discussed.

Over-expression and refolding of isotopically labeled recombinant catalytic domain of human macrophage elastase (MMP-12) for NMR studies.

Human macrophage elastase (MMP-12) plays an important role in inflammatory processes and is involved in a number of physiological or pathological situations, such as conversion of plasminogen into angiostatin, allergic airway inflammation, vascular remodeling or alteration, as well as emphysema, and has been justified as a novel drug target. Here, we report the over-expression in Escherichia coil, purification and refolding of MMP-12 catalytic domain for NMR studies. The primary sequence of expressed protein was identified by means of MALDI-TOF MS, and was confirmed by the MALDI-TOF MS data of trypsin-digested peptides. A significantly optimized protocol has been worked out to prepare 15N and/or 13C-labeled MMP-12 catalytic domain, and the yield of the purified protein is estimated to 10-12 mg from 0.5L of M9 minimal media. Finally, the 15N-1H HSQC spectrum of uniformly 15N-labeled MMP-12 catalytic domain indicates the presence of well-ordered and properly folded protein in a monomeric form.

NMR solution structure of BmK-betaIT, an excitatory scorpion beta-toxin without a 'hot spot' at the relevant position.

BmK-betaIT (previously named as Bm32-VI in the literature), an excitatory scorpion beta-toxin, is purified from the venom of the Chinese scorpion Buthus martensii Karsch. It features a primary sequence typical of the excitatory anti-insect toxins: two contiguous Cys residues (Cys37-Cys38) and a shifted location of the fourth disulfide bridges (Cys38-Cys64), and demonstrates bioactivity characteristic of the excitatory beta-toxins. However, it is noteworthy that BmK-betaIT is not conserved with a glutamate residue at the preceding position of the third Cys residue, and is the first example having a non-glutamate residue at the relevant position in the excitatory scorpion beta-toxin subfamily. The 3D structure of BmK-betaIT is determined with 2D NMR spectroscopy and molecular modeling. The solution structure of BmK-betaIT is closely similar to those of BmK IT-AP and Bj-xtrIT, only distinct from the latter by lack of an alpha(0)-helix. The surface functional patch comparison with those of BmK IT-AP and Bj-xtrIT reveals their striking similarity in the spatial arrangement. These results infer that the functional surface of beta-toxins is composed of two binding regions and a functional site. The main binding site is consisted of hydrophobic residues surrounding the alpha(1)-helix and its preceding loop, which is common to all beta-type scorpion toxins affecting Na(+) channels. The second binding site, which determines the specificity of the toxin, locates at the C-terminus for excitatory insect beta-toxin, while rests at the beta-sheet and its linking loop for anti-mammal toxins. The functional site involved in the voltage sensor-trapping model, which characterizes the function of all beta-toxins, is the negatively charged residue Glu15.

The expression and refolding of isotopically labeled recombinant Matrilysin for NMR studies.

Matrilysin (MMP7) is the smallest member of matrix metalloproteinases (MMPs) family, which are collectively responsible for remodeling of connective tissue. MMP7 plays an essential role in cancer, innate immunity, and in inflammatory disorders, and has been justified as a novel drug target. Here, we report the gene synthesis, overexpression in Escherichia coli, purification and refolding of MMP7. The gene of Matrilysin was synthesized based on PCR method and overexpressed in E. coli in the form of inclusion bodies. The protein was subsequently purified and refolded to yield sufficient quantities for structural and functional studies. The purified protein was characterized by means of MALDI-TOF mass spectroscopy and dynamic light scattering (DLS) analysis. The MS data confirms the correctness of the primary sequence, while DLS experiment proves that the protein exists as a monomeric form. A significantly optimized protocol has been worked out to prepare (15)N and/or (13)C-labeled MMP7 in minimal medium with high yields for NMR studies. Under the various conditions optimized for the purification of MMP7, the yield of the purified protein is estimated to be 18-20 mg from 0.5 L of M9 minimal media. Finally, the (15)N-1H HSQC spectrum of uniformly (15)N-labeled MMP7 sample reveals that the protein is properly folded, and exists in a well-ordered structure.