Site-Specific Structural Variations Accompanying Tubular Assembly of the HIV-1 Capsid Protein

The 231-residue capsid (CA) protein of human immunodeficiency virus type 1 (HIV-1) spontaneously self-assembles into tubes with a hexagonal lattice that is believed to mimic the surface lattice of conical capsid cores within intact virions. We report the results of solid-state nuclear magnetic resonance (NMR) measurements on HIV-1 CA tubes that provide new information regarding changes in molecular structure that accompany CA self-assembly, local dynamics within CA tubes, and possible mechanisms for the generation of lattice curvature. This information is contained in site-specific assignments of signals in two- and three-dimensional solid-state NMR spectra, conformation-dependent ¹⁵N and ¹³C NMR chemical shifts, detection of highly dynamic residues under solution NMR conditions, measurements of local variations in transverse spin relaxation rates of amide ¹H nuclei, and quantitative measurements of site-specific ¹⁵N–¹⁵N dipole–dipole couplings. Our data show that most of the CA sequence is conformationally ordered and relatively rigid in tubular assemblies and that structures of the N-terminal domain (NTD) and the C-terminal domain (CTD) observed in solution are largely retained. However, specific segments, including the N-terminal β-hairpin, the cyclophilin A binding loop, the inter-domain linker, segments involved in intermolecular NTD–CTD interactions, and the C-terminal tail, have substantial static or dynamical disorder in tubular assemblies. Other segments, including the 3₁₀-helical segment in CTD, undergo clear conformational changes. Structural variations associated with curvature of the CA lattice appear to be localized in the inter-domain linker and intermolecular NTD–CTD interface, while structural variations within NTD hexamers, around local 3-fold symmetry axes, and in CTD–CTD dimerization interfaces are less significant.     

Segmental isotopic labeling of HIV-1 capsid protein assemblies for solid state NMR

Recent studies of noncrystalline HIV-1 capsid protein (CA) assemblies by our laboratory and by Polenova and coworkers (Protein Sci 19:716–730, 2010; J Mol Biol 426:1109–1127, 2014; J Biol Chem 291:13098–13112, 2016; J Am Chem Soc 138:8538–8546, 2016; J Am Chem Soc 138:12029–12032, 2016; J Am Chem Soc 134:6455–6466, 2012; J Am Chem Soc 132:1976–1987, 2010; J Am Chem Soc 135:17793–17803, 2013; Proc Natl Acad Sci USA 112:14617–14622, 2015; J Am Chem Soc 138:14066–14075, 2016) have established the capability of solid state nuclear magnetic resonance (NMR) measurements to provide site-specific structural and dynamical information that is not available from other types of measurements. Nonetheless, the relatively high molecular weight of HIV-1 CA leads to congestion of solid state NMR spectra of fully isotopically labeled assemblies that has been an impediment to further progress. Here we describe an efficient protocol for production of segmentally labeled HIV-1 CA samples in which either the N-terminal domain (NTD) or the C-terminal domain (CTD) is uniformly ¹⁵N,¹³C-labeled. Segmental labeling is achieved by trans-splicing, using the DnaE split intein. Comparisons of two-dimensional solid state NMR spectra of fully labeled and segmentally labeled tubular CA assemblies show substantial improvements in spectral resolution. The molecular structure of HIV-1 assemblies is not significantly perturbed by the single Ser-to-Cys substitution that we introduce between NTD and CTD segments, as required for trans-splicing.     

Conformation and dynamics changes of bacteriorhodopsin and its D85N mutant in the absence of 2D crystalline lattice as revealed by site-directed C NMR

¹³C NMR spectra of [3-¹³C]Ala- and [1-¹³C]Val-labeled D85N mutant of bacteriorhodopsin (bR) reconstituted in egg PC or DMPC bilayers were recorded to gain insight into their secondary structures and dynamics. They were substantially suppressed as compared with those of 2D crystals, especially at the loops and several transmembrane α_II-helices. Surprisingly, the ¹³C NMR spectra of [3-¹³C]Ala-D85N turned out to be very similar to those of [3-¹³C]Ala-bR in lipid bilayers, in spite of the presence of globular conformational and dynamics changes in the former as found from 2D crystalline preparations. No further spectral change was also noted between the ground (pH 7) and M-like state (pH 10) as far as D85N in lipid bilayers was examined, in spite of their distinct changes in the 2D crystalline state. This is mainly caused by that the resulting ¹³C NMR peaks which are sensitive to conformation and dynamics changes in the loops and several transmembrane α_II-helices of the M-like state are suppressed already by fluctuation motions in the order of 10⁴-10⁵ Hz interfered with frequencies of magic angle spinning or proton decoupling. However, ¹³C NMR signal from the cytoplasmic α-helix protruding from the membrane surface is not strongly influenced by 2D crystal or monomer. Deceptively simplified carbonyl ¹³C NMR signals of the loop and transmembrane α-helices followed by Pro residues in [1-¹³C]Val-labeled bR and D85N in 2D crystal are split into two peaks for reconstituted preparations in the absence of 2D crystalline lattice. Fortunately, ¹³C NMR spectral feature of reconstituted [1-¹³C]Val and [3-¹³C]Ala-labeled bR and D85N was recovered to yield characteristic feature of 2D crystalline form in gel-forming lipids achieved at lowered temperatures.     

1H, 13C, and 15N resonance assignment of the N-terminal domain of Mason-Pfizer monkey virus capsid protein, CA 1-140

Mason-Pfizer monkey virus (M-PMV) belongs to the family of betaretroviruses characterized by the assembly of immature particles within cytoplasm of infected cells in contrast to other retroviruses (e.g. HIV, RSV) that assemble their immature particles at a plasma membrane. Simultaneously with or shortly after budding a virus-encoded protease is activated and the Gag polyprotein is cleaved into three major structural proteins: matrix (MA), capsid (CA), and nucleocapsid (NC) protein. Mature retroviral CA proteins consist of two independently folded structural domains: N-terminal domain (NTD) and C-terminal dimerization domain (CTD), separated by a flexible linker. As a first step toward the solution structure elucidation, we present nearly complete backbone and side-chain ¹H, ¹⁵N and ¹³C resonance assignment of the M-PMV NTD CA.     

Synthetic OCP heterodimers are photoactive and recapitulate the fusion of two primitive carotenoproteins in the evolution of cyanobacterial photoprotection

The orange carotenoid protein (OCP) governs photoprotection in the majority of cyanobacteria. It is structurally and functionally modular, comprised of a C‐terminal regulatory domain (CTD), an N‐terminal effector domain (NTD) and a ketocarotenoid; the chromophore spans the two domains in the ground state and translocates fully into the NTD upon illumination. Using both the canonical OCP1 from Fremyella diplosiphon and the presumably more primitive OCP2 paralog from the same organism, we show that an NTD‐CTD heterodimer forms when the domains are expressed as separate polypeptides. The carotenoid is required for the heterodimeric association, assembling an orange complex which is stable in the dark. Both OCP1 and OCP2 heterodimers are photoactive, undergoing light‐driven heterodimer dissociation, but differ in their ability to reassociate in darkness, setting the stage for bioengineering photoprotection in cyanobacteria as well as for developing new photoswitches for biotechnology. Additionally, we reveal that homodimeric CTD can bind carotenoid in the absence of NTD, and name this truncated variant the C‐terminal domain‐like carotenoid protein (CCP). This finding supports the hypothesis that the OCP evolved from an ancient fusion event between genes for two different carotenoid‐binding proteins ancestral to the NTD and CTD. We suggest that the CCP and its homologs constitute a new family of carotenoproteins within the NTF2‐like superfamily found across all kingdoms of life.     

Structures and dynamics of hibernating ribosomes from Staphylococcus aureus mediated by intermolecular interactions of HPF

In bacteria, ribosomal hibernation shuts down translation as a response to stress, through reversible binding of stress‐induced proteins to ribosomes. This process typically involves the formation of 100S ribosome dimers. Here, we present the structures of hibernating ribosomes from human pathogen Staphylococcus aureus containing a long variant of the hibernation‐promoting factor (SaHPF) that we solved using cryo‐electron microscopy. Our reconstructions reveal that the N‐terminal domain (NTD) of SaHPF binds to the 30S subunit as observed for shorter variants of HPF in other species. The C‐terminal domain (CTD) of SaHPF protrudes out of each ribosome in order to mediate dimerization. Using NMR, we characterized the interactions at the CTD‐dimer interface. Secondary interactions are provided by helix 26 of the 16S ribosomal RNA. We also show that ribosomes in the 100S particle adopt both rotated and unrotated conformations. Overall, our work illustrates a specific mode of ribosome dimerization by long HPF, a finding that may help improve the selectivity of antimicrobials.     

Global fold of human cannabinoid type 2 receptor probed by solid‐state 13C‐, 15N‐MAS NMR and molecular dynamics simulations

The global fold of human cannabinoid type 2 (CB₂) receptor in the agonist‐bound active state in lipid bilayers was investigated by solid‐state ¹³C‐ and ¹⁵N magic‐angle spinning (MAS) NMR, in combination with chemical‐shift prediction from a structural model of the receptor obtained by microsecond‐long molecular dynamics (MD) simulations. Uniformly ¹³C‐ and ¹⁵N‐labeled CB₂ receptor was expressed in milligram quantities by bacterial fermentation, purified, and functionally reconstituted into liposomes. ¹³C MAS NMR spectra were recorded without sensitivity enhancement for direct comparison of C_α, C_β, and C?O bands of superimposed resonances with predictions from protein structures generated by MD. The experimental NMR spectra matched the calculated spectra reasonably well indicating agreement of the global fold of the protein between experiment and simulations. In particular, the ¹³C chemical shift distribution of C_α resonances was shown to be very sensitive to both the primary amino acid sequence and the secondary structure of CB₂. Thus the shape of the C_α band can be used as an indicator of CB₂ global fold. The prediction from MD simulations indicated that upon receptor activation a rather limited number of amino acid residues, mainly located in the extracellular Loop 2 and the second half of intracellular Loop 3, change their chemical shifts significantly (≥1.5 ppm for carbons and ≥5.0 ppm for nitrogens). Simulated two‐dimensional ¹³C_α(i)? ¹³C?O(i) and ¹³C?O(i)? ¹⁵NH(i + 1) dipolar‐interaction correlation spectra provide guidance for selective amino acid labeling and signal assignment schemes to study the molecular mechanism of activation of CB₂ by solid‐state MAS NMR. Proteins 2014; 82:452–465. © 2013 Wiley Periodicals, Inc.     

1H, 13C and 15N chemical shift assignments for the N-terminal domain of the voltage-gated potassium channel-hERG

The human ether à go-go related gene (hERG) voltage-gated potassium controls the rapid delayed rectifier potassium current (I_ks) in heart. The N-terminal 135 amino acids (NTD) form a Per-Arnt-Sim (PAS) domain which involves in signal transduction and protein–protein interactions. NTD was shown to be necessary for the regulation of the channel activity through its interaction with the channel pore region of hERG. Mutations in NTD were related to serious heart diseases. We report the ¹H, ¹³C and ¹⁵N chemical shift assignments for NTD using 2D and 3D heteronuclear NMR experiments. More than 95% backbone resonance assignments were obtained.     

Conformational analysis of the full‐length M2 protein of the influenza A virus using solid‐state NMR

The influenza A M2 protein forms a proton channel for virus infection and mediates virus assembly and budding. While extensive structural information is known about the transmembrane helix and an adjacent amphipathic helix, the conformation of the N‐terminal ectodomain and the C‐terminal cytoplasmic tail remains largely unknown. Using two‐dimensional (2D) magic‐angle‐spinning solid‐state NMR, we have investigated the secondary structure and dynamics of full‐length M2 (M2FL) and found them to depend on the membrane composition. In 2D ¹³C DARR correlation spectra, 1,2‐dimyristoyl‐sn‐glycero‐3‐phosphocholine (DMPC)‐bound M2FL exhibits several peaks at β‐sheet chemical shifts, which result from water‐exposed extramembrane residues. In contrast, M2FL bound to cholesterol‐containing membranes gives predominantly α‐helical chemical shifts. Two‐dimensional J‐INADEQUATE spectra and variable‐temperature ¹³C spectra indicate that DMPC‐bound M2FL is highly dynamic while the cholesterol‐containing membranes significantly immobilize the protein at physiological temperature. Chemical‐shift prediction for various secondary‐structure models suggests that the β‐strand is located at the N‐terminus of the DMPC‐bound protein, while the cytoplasmic domain is unstructured. This prediction is confirmed by the 2D DARR spectrum of the ectodomain‐truncated M2(21–97), which no longer exhibits β‐sheet chemical shifts in the DMPC‐bound state. We propose that the M2 conformational change results from the influence of cholesterol, and the increased helicity of M2FL in cholesterol‐rich membranes may be relevant for M2 interaction with the matrix protein M1 during virus assembly and budding. The successful determination of the β‐strand location suggests that chemical‐shift prediction is a promising approach for obtaining structural information of disordered proteins before resonance assignment.     

Function and solution structure of the Arabidopsis thaliana RALF8 peptide

We report the recombinant preparation from Escherichia coli cells of samples of two closely related, small, secreted cysteine‐rich plant peptides: rapid alkalinization factor 1 (RALF1) and rapid alkalinization factor 8 (RALF8). Purified samples of the native sequence of RALF8 exhibited well‐resolved nuclear magnetic resonance (NMR) spectra and also biological activity through interaction with a plant receptor kinase, cytoplasmic calcium mobilization, and in vivo root growth suppression. By contrast, RALF1 could only be isolated from inclusion bodies as a construct containing an N‐terminal His‐tag; its poorly resolved NMR spectrum was indicative of aggregation. We prepared samples of the RALF8 peptide labeled with ¹⁵N and ¹³C for NMR analysis and obtained near complete ¹H, ¹³C, and ¹⁵N NMR assignments; determined the disulfide pairing of its four cysteine residues; and examined its solution structure. RALF8 is mostly disordered except for the two loops spanned by each of its two disulfide bridges.     

1H, 15N and 13C assignments of the dimeric C-terminal domain of HIV-1 capsid protein

HIV-1 capsid protein (CA) encloses the viral RNA genome and forms a conical-shaped particle in the mature HIV-1 virion, with orderly capsid assembly and disassembly critically important for viral infectivity. The 231 residue CA is composed of two helical domains, connected by a short linker sequence. In solution, CA exhibits concentration dependent dimerization which is mediated by the C-terminal domain (CTD). Here, we present nearly complete ¹H, ¹⁵N and ¹³C assignments for the 20 kDa homodimeric CA–CTD, a prerequisite for structural characterization of the CA–CTD dimer.     

Resonance assignment of 13C/15N labeled solid proteins by two- and three-dimensional magic-angle-spinning NMR

The comprehensive structure determination of isotopically labeled proteins by solid-state NMR requires sequence-specific assignment of ¹³C and ¹⁵ N spectra. We describe several 2D and 3D MAS correlation techniques for resonance assignment and apply them, at 7.0 Tesla, to ¹³C and ¹⁵N labeled ubiquitin to examine the extent of resonance assignments in the solid state. Both interresidue and intraresidue assignments of the ¹³C and ¹⁵N resonances are addressed. The interresidue assignment was carried out by an N(CO)CA technique, which yields N_i-C_i–1 connectivities in protein backbones via two steps of dipolar-mediated coherence transfer. The intraresidue connectivities were obtained from a new 3D NCACB technique, which utilizes the well resolved C chemical shift to distinguish the different amino acids. Additional amino acid type assignment was provided by a ¹³C spin diffusion experiment, which exhibits ¹³C spin pairs as off-diagonal intensities in the 2D spectrum. To better resolve carbons with similar chemical shifts, we also performed a dipolar-mediated INADEQUATE experiment. By cross-referencing these spectra and exploiting the selective and extensive ¹³ C labeling approach, we assigned 25% of the amino acids in ubiquitin sequence-specifically and 47% of the residues to the amino acid types. The sensitivity and resolution of these experiments are evaluated, especially in the context of the selective and extensive ¹³C labeling approach.     

Second-site compensatory mutations of HIV-1 capsid mutations

The human immunodeficiency virus (HIV) capsid (CA) protein assembles into a hexameric lattice that forms the mature virus core. Contacts between the CA N-terminal domain (NTD) of one monomer and the C-terminal domain (CTD) of the adjacent monomer are important for the assembly of this core. In this study, we have examined the effects of mutations in the NTD region associated with this interaction. We have found that such mutations yielded modest reductions of virus release but major effects on viral infectivity. Cell culture and in vitro assays indicate that the infectivity defects relate to abnormalities in the viral cores. We have selected second-site compensatory mutations that partially restored HIV infectivity. These mutations map to the CA CTD and to spacer peptide 1 (SP1), the portion of the precursor Gag protein immediately C terminal to the CTD. The compensatory mutations do not locate to the molecularly modeled intermolecular NTD-CTD interface. Rather, the compensatory mutations appear to act indirectly, possibly by realignment of the C-terminal helix of the CA CTD, which participates in the NTD-CTD interface and has been shown to serve an important role in the assembly of infectious virus.     

X‐ray crystal structure of the N‐terminal region of Moloney murine leukemia virus integrase and its implications for viral DNA recognition

The retroviral integrase (IN) carries out the integration of a dsDNA copy of the viral genome into the host DNA, an essential step for viral replication. All IN proteins have three general domains, the N‐terminal domain (NTD), the catalytic core domain, and the C‐terminal domain. The NTD includes an HHCC zinc finger‐like motif, which is conserved in all retroviral IN proteins. Two crystal structures of Moloney murine leukemia virus (M‐MuLV) IN N‐terminal region (NTR) constructs that both include an N‐terminal extension domain (NED, residues 1–44) and an HHCC zinc‐finger NTD (residues 45–105), in two crystal forms are reported. The structures of IN NTR constructs encoding residues 1–105 (NTR_1–105) and 8–105 (NTR_8–105) were determined at 2.7 and 2.15 Å resolution, respectively and belong to different space groups. While both crystal forms have similar protomer structures, NTR_1–105 packs as a dimer and NTR_8–105 packs as a tetramer in the asymmetric unit. The structure of the NED consists of three anti‐parallel β‐strands and an α‐helix, similar to the NED of prototype foamy virus (PFV) IN. These three β‐strands form an extended β‐sheet with another β‐strand in the HHCC Zn²⁺ binding domain, which is a unique structural feature for the M‐MuLV IN. The HHCC Zn²⁺ binding domain structure is similar to that in HIV and PFV INs, with variations within the loop regions. Differences between the PFV and MLV IN NEDs localize at regions identified to interact with the PFV LTR and are compared with established biochemical and virological data for M‐MuLV. Proteins 2017; 85:647–656. © 2016 Wiley Periodicals, Inc.     

Partial site-specific assignment of a uniformly C, N enriched membrane protein, light-harvesting complex 1 (LH1), by solid state NMR

Partial site-specific assignments are reported for the solid state NMR spectra of light-harvesting complex 1, a 160 kDa integral membrane protein. The assignments were derived from 600 MHz ¹⁵N-¹³CO-¹³Cα and ¹⁵N-¹³Cα-¹³CX correlation spectra, using uniformly ¹³C, ¹⁵N enriched hydrated material, in an intact and precipitated form. Sequential assignments were verified using characteristic ¹⁵N-¹³Cα-¹³Cβ side chain chemical shifts observed in 3D experiments. Tertiary contacts found in 2D DARR spectra of the selectively ¹³C enriched sample provided further confirmatory evidence for the assignments. The assignments include the region of the Histidine ligands binding the Bacteriochlorophyll chromophore. The chemical shifts of Cα and Cβ resonances indicated the presence of typical α-helical secondary structure, consistent with previous studies.     

NMR characterization of an engineered domain fusion between maltose binding protein and TEM1 β‐lactamase provides insight into its structure and allosteric mechanism

RG13 is a 72 kDa engineered allosteric enzyme comprised of a fusion between maltose binding protein (MBP) and TEM1 β‐lactamase (BLA) for which maltose is a positive effector of BLA activity. We have used NMR spectroscopy to acquire [¹⁵N, ¹H]‐TROSY‐HSQC spectra of RG13 in the presence and absence of maltose. The RG13 chemical shift data was compared to the published chemical shift data of MBP and BLA. The spectra are consistent with the expectation that the individual domain structures of RG13 are substantially conserved from MBP and BLA. Differences in the spectra are consistent with the fusion geometry of MBP and BLA and the maltose‐dependent differences in the kinetics of RG13 enzyme activity. In particular, the spectra provide evidence for a maltose‐dependent conformational change of a key active site glutamate involved in deacylation of the enzyme‐substrate intermediate. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Secondary structural analysis of proteins based on 13C chemical shift assignments in unresolved solid-state NMR spectra enhanced by fragmented structure database

Magic-angle-spinning solid-state ¹³C NMR spectroscopy is useful for structural analysis of non-crystalline proteins. However, the signal assignments and structural analysis are often hampered by the signal overlaps primarily due to minor structural heterogeneities, especially for uniformly-¹³C,¹⁵N labeled samples. To overcome this problem, we present a method for assigning ¹³C chemical shifts and secondary structures from unresolved two-dimensional ¹³C–¹³C MAS NMR spectra by spectral fitting, named reconstruction of spectra using protein local structures (RESPLS). The spectral fitting was conducted using databases of protein fragmented structures related to ¹³C^α, ¹³C^β, and ¹³C′ chemical shifts and cross-peak intensities. The experimental ¹³C–¹³C inter- and intra-residue correlation spectra of uniformly isotope-labeled ubiquitin in the lyophilized state had a few broad peaks. The fitting analysis for these spectra provided sequence-specific C^α, C^β, and C′ chemical shifts with an accuracy of about 1.5 ppm, which enabled the assignment of the secondary structures with an accuracy of 79 %. The structural heterogeneity of the lyophilized ubiquitin is revealed from the results. Test of RESPLS analysis for simulated spectra of five different types of proteins indicated that the method allowed the secondary structure determination with accuracy of about 80 % for the 50–200 residue proteins. These results demonstrate that the RESPLS approach expands the applicability of the NMR to non-crystalline proteins exhibiting unresolved ¹³C NMR spectra, such as lyophilized proteins, amyloids, membrane proteins and proteins in living cells.     

Residue-specific millisecond to microsecond fluctuations in bacteriorhodopsin induced by disrupted or disorganized two-dimensional crystalline lattice, through modified lipid-helix and helix-helix interactions, as revealed by C NMR

We have recorded ¹³C NMR spectra of [3-¹³C]-, [1-¹³C]Ala-, and [1-¹³C]Val-labeled bacteriorhodopsin (bR), W80L and W12L mutants and bacterio-opsin (bO) from retinal-deficient E1001 strain, in order to examine the possibility of their millisecond to microsecond local fluctuations with correlation time in the order of 10⁻⁴ to 10⁻⁵ s, induced or prevented by disruption or assembly of two-dimensional (2D) crystalline lattice, respectively, at ambient temperature. The presence of disrupted or disorganized 2D lattice for W12L, W80L and bO from E1001 strain was readily visualized by increased relative proportions of surrounding lipids per protein, together with their broadened ¹³C NMR signals of transmembrane α-helices and loops in [3-¹³C]Ala-labeled proteins, with reference to those of wild-type. In contrast, ¹³C CP-MAS NMR spectra of [1-¹³C]Ala- and Val-labeled these mutants were almost completely suppressed, owing to the presence of fluctuations with time scale of 10⁻⁴ s interfered with magic angle spinning. In particular, ¹³C NMR signals of [1-¹³C]Ala-labeled transmembrane α-helices of wild-type were almost completely suppressed at the interface between the surface and inner part (up to 8.7 Å deep from the surface) with reference to those of the similarly suppressed peaks by Mn²⁺-induced accelerated spin-spin relaxation rate. Such fluctuation-induced suppression of ¹³C NMR peaks from the interfacial regions, however, was less significant for [1-¹³C]Val-labeled proteins, because fluctuation motions in Val residues with bulky side-chains at the C_α moiety were modified to those of longer correlation time (>10⁻⁴ s), if any, by residue-specific manner. To support this view, we found that such suppressed ¹³C NMR signals of [1-¹³C]Ala-labeled peaks in the wild-type were recovered for D85N and bO in which correlation times of fluctuations were shifted to the order of 10⁻⁵ s due to modified helix-helix interactions as previously pointed out [Biochemistry, 39 (2000) 14472; J. Biochem. (Tokyo) 127 (2000) 861].