Solution-state NMR assignment and secondary structure propensity of the full length and minimalistic-truncated prefibrillar monomeric form of biofilm forming functional amyloid FapC from Pseudomonas aeruginosa.

Functional bacterial amyloids provide structural scaffolding to bacterial biofilms. In contrast to the pathological amyloids, they have a role in vivo and are tightly regulated. Their presence is essential to the integrity of the bacterial communities surviving in biofilms and may cause serious health complications. Targeting amyloids in biofilms could be a novel approach to prevent chronic infections. However, structural information is very scarce on them in both soluble monomeric and insoluble fibrillar forms, hindering our molecular understanding and strategies to fight biofilm related diseases. Here, we present solution-state NMR assignment of 250 amino acid long biofilm-forming functional-amyloid FapC from Pseudomonas aeruginosa. We studied full-length (FL) and shorter minimalistic-truncated (L2R3C) FapC constructs without the signal-sequence that is required for secretion. 91% and 100% backbone NH resonance assignments for FL and L2R3C constructs, respectively, indicate that soluble monomeric FapC is predominantly disordered, with sizeable secondary structural propensities mostly as PP2 helices, but also as α-helices and ß-sheets highlighting hotspots for fibrillation initiation interface. A shorter construct showing almost identical NMR chemical shifts highlights the promise of utilizing it for more demanding solid-state NMR studies that require methods to alleviate signal redundancy due to almost identical repeat units. This study provides key NMR resonance assignments for future structural studies of soluble, pre-fibrillar and fibrillar forms of FapC.

The Magic of Linking Rings: Discovery of a Unique Photoinduced Fluorescent Protein Crosslink.

Fluorosubstituted tryptophans serve as valuable probes for fluorescence and nuclear magnetic resonance (NMR) studies of proteins. Here, we describe an unusual photoreactivity introduced by replacing the single tryptophan in cyclophilin A with 7-fluoro-tryptophan. UV exposure at 282 nm defluorinates 7-fluoro-tryptophan and crosslinks it to a nearby phenylalanine, generating a bright fluorophore. The crosslink-containing fluorescent protein possesses a large quantum yield of ∼0.40 with a fluorescence lifetime of 2.38 ns. The chemical nature of the crosslink and the three-dimensional protein structure were determined by mass spectrometry and NMR spectroscopy. To the best of our knowledge, this is the first report of a Phe-Trp crosslink in a protein. Our finding may break new ground for developing novel fluorescence probes and for devising new strategies to exploit aromatic crosslinks in proteins.

Structure of HIV-1 Vpr in complex with the human nucleotide excision repair protein hHR23A.

HIV-1 Vpr is a prototypic member of a large family of structurally related lentiviral virulence factors that antagonize various aspects of innate antiviral immunity. It subverts host cell DNA repair and protein degradation machineries by binding and inhibiting specific post-replication repair enzymes, linking them via the DCAF1 substrate adaptor to the Cullin 4 RING E3 ligase (CRL4DCAF1). HIV-1 Vpr also binds to the multi-domain protein hHR23A, which interacts with the nucleotide excision repair protein XPC and shuttles ubiquitinated proteins to the proteasome. Here, we report the atomic resolution structure of Vpr in complex with the C-terminal half of hHR23A, containing the XPC-binding (XPCB) and ubiquitin-associated (UBA2) domains. The XPCB and UBA2 domains bind to different sides of Vpr's 3-helix-bundle structure, with UBA2 interacting with the α2 and α3 helices of Vpr, while the XPCB domain contacts the opposite side of Vpr's α3 helix. The structure as well as biochemical results reveal that hHR23A and DCAF1 use overlapping binding surfaces on Vpr, even though the two proteins exhibit entirely different three-dimensional structures. Our findings show that Vpr independently targets hHR23A- and DCAF1- dependent pathways and highlight HIV-1 Vpr as a versatile module that interferes with DNA repair and protein degradation pathways.

Complete 1H, 13C, 15N resonance assignments and secondary structure of the Vpr binding region of hHR23A (residues 223-363).

Comprehensive resonance assignments and delineation of the secondary structure elements of the C-terminal Vpr-binding region of hHR23A, residues 223-363, were achieved by triple-resonance NMR experiments on uniformly 13C,15N-labeled protein. Assignments are 100% and > 95% complete for backbone and side-chain resonances, respectively. This data constitutes important complementary information for our ongoing structure determination of the Vpr-hHR23A(223-363) complex. At high concentrations, severe line-broadening was observed for several residues in the 1H-15N HSQC spectrum, most likely resulting from inter-molecular interactions.

Dynamic Nuclear Polarization Magic-Angle Spinning Nuclear Magnetic Resonance Combined with Molecular Dynamics Simulations Permits Detection of Order and Disorder in Viral Assemblies.

We report dynamic nuclear polarization (DNP)-enhanced magic-angle spinning (MAS) NMR spectroscopy in viral capsids from HIV-1 and bacteriophage AP205. Viruses regulate their life cycles and infectivity through modulation of their structures and dynamics. While static structures of capsids from several viruses are now accessible with near-atomic-level resolution, atomic-level understanding of functionally important motions in assembled capsids is lacking. We observed up to 64-fold signal enhancements by DNP, which permitted in-depth analysis of these assemblies. For the HIV-1 CA assemblies, a remarkably high spectral resolution in the 3D and 2D heteronuclear data sets permitted the assignment of a significant fraction of backbone and side-chain resonances. Using an integrated DNP MAS NMR and molecular dynamics (MD) simulation approach, the conformational space sampled by the assembled capsid at cryogenic temperatures was mapped. Qualitatively, a remarkable agreement was observed for the experimental 13C/15N chemical shift distributions and those calculated from substructures along the MD trajectory. Residues that are mobile at physiological temperatures are frozen out in multiple conformers at cryogenic conditions, resulting in broad experimental and calculated chemical shift distributions. Overall, our results suggest that DNP MAS NMR measurements in combination with MD simulations facilitate a thorough understanding of the dynamic signatures of viral capsids.

Functional analysis of the secondary HIV-1 capsid binding site in the host protein cyclophilin A.

BACKGROUND: Efficient HIV-1 replication depends on interaction of the viral capsid with the host protein cyclophilin A (CypA). CypA, a peptidylprolyl isomerase, binds to an exposed loop in the viral CA protein via the enzyme's active site. Recent structural analysis of CypA in complex with CA tubes in conjunction with molecular dynamics simulations identified a secondary CA binding site on CypA that allows a bridging interaction with two hexameric subunits of the assembled CA lattice, leading to capsid stabilization (Liu et al. in Nat Commun 7:10714, 2016). RESULTS: We performed mutational analysis of residues that have been proposed to mediate CA binding at the secondary binding site on CypA (A25, K27, P29 and K30) and tested the effects of the amino acid substitutions using interaction assays and HIV-1 infection assays in cells. The binding of recombinant CypA to self-assembled CA tubes or native HIV-1 capsids was measured in vitro using a quantitative fluorescence microscopy binding assay revealing that affinity and stoichiometry of CypA to the CA lattice was not affected by the substitutions. To test for functionality of the CypA secondary CA-binding site in HIV-1 infection, mutant CypA proteins were expressed in cells in which endogenous CypA was deleted, and the effects on HIV-1 infection were assayed. In normal HeLa-P4 cells, infection with HIV-1 bearing the A92E substitution in CA is inhibited by endogenous CypA and was inhibited to the same extent by expression of CypA mutants in CypA-null HeLa-P4 cells. Expression of the mutant CypA proteins in CypA-null Jurkat cells restored their permissiveness to infection by wild type HIV-1. CONCLUSIONS: The amino acid changes at A25, K27, P29 and K30 did not affect the affinity of CypA for the CA lattice and did not impair CypA function in infection assays suggesting that these residues are not part of a secondary CA binding site on CypA.

Fast Magic-Angle Spinning 19 F†NMR Spectroscopy of HIV-1 Capsid Protein Assemblies.

19 F NMR spectroscopy is an attractive and growing area of research with broad applications in biochemistry, chemical biology, medicinal chemistry, and materials science. We have explored fast magic angle spinning (MAS) 19 F solid-state NMR spectroscopy in assemblies of HIV-1 capsid protein. Tryptophan residues with fluorine substitution at the 5-position of the indole ring were used as the reporters. The 19 F chemical shifts for the five tryptophan residues are distinct, reflecting differences in their local environment. Spin-diffusion and radio-frequency-driven-recoupling experiments were performed at MAS frequencies of 35 kHz and 40-60 kHz, respectively. Fast MAS frequencies of 40-60 kHz are essential for consistently establishing 19 F-19 F correlations, yielding interatomic distances of the order of 20 Å. Our results demonstrate the potential of fast MAS 19 F NMR spectroscopy for structural analysis in large biological assemblies.

Correction to: NMR structure of the HIV-1 reverse transcriptase thumb subdomain.

In the original publication of the article, the given name and family name of the author P. Andrew Karplus was published incorrectly. The name should read as "P. Andrew" - Given name and "Karplus" - Family name.

CryoEM Structure Refinement by Integrating NMR Chemical Shifts with Molecular Dynamics Simulations.

Single particle cryoEM has emerged as a powerful method for structure determination of proteins and complexes, complementing X-ray crystallography and NMR spectroscopy. Yet, for many systems, the resolution of cryoEM density map has been limited to 4-6 Å, which only allows for resolving bulky amino acids side chains, thus hindering accurate model building from the density map. On the other hand, experimental chemical shifts (CS) from solution and solid state MAS NMR spectra provide atomic level data for each amino acid within a molecule or a complex; however, structure determination of large complexes and assemblies based on NMR data alone remains challenging. Here, we present a novel integrated strategy to combine the highly complementary experimental data from cryoEM and NMR computationally by molecular dynamics simulations to derive an atomistic model, which is not attainable by either approach alone. We use the HIV-1 capsid protein (CA) C-terminal domain as well as the large capsid assembly to demonstrate the feasibility of this approach, termed NMR CS-biased cryoEM structure refinement.

The Histone Modification Domain of Paf1 Complex Subunit Rtf1 Directly Stimulates H2B Ubiquitylation through an Interaction with Rad6.

The five-subunit yeast Paf1 complex (Paf1C) regulates all stages of transcription and is critical for the monoubiquitylation of histone H2B (H2Bub), a modification that broadly influences chromatin structure and eukaryotic transcription. Here, we show that the histone modification domain (HMD) of Paf1C subunit Rtf1 directly interacts with the ubiquitin conjugase Rad6 and stimulates H2Bub independently of transcription. We present the crystal structure of the Rtf1 HMD and use site-specific, in vivo crosslinking to identify a conserved Rad6 interaction surface. Utilizing ChIP-exo analysis, we define the localization patterns of the H2Bub machinery at high resolution and demonstrate the importance of Paf1C in targeting the Rtf1 HMD, and thereby H2Bub, to its appropriate genomic locations. Finally, we observe HMD-dependent stimulation of H2Bub in a transcription-free, reconstituted in vitro system. Taken together, our results argue for an active role for Paf1C in promoting H2Bub and ensuring its proper localization in vivo.

NMR structure of the HIV-1 reverse transcriptase thumb subdomain.

The solution NMR structure of the isolated thumb subdomain of HIV-1 reverse transcriptase (RT) has been determined. A detailed comparison of the current structure with dozens of the highest resolution crystal structures of this domain in the context of the full-length enzyme reveals that the overall structures are very similar, with only two regions exhibiting local conformational differences. The C-terminal capping pattern of the αH helix is subtly different, and the loop connecting the αI and αJ helices in the p51 chain of the full-length p51/p66 heterodimeric RT differs from our NMR structure due to unique packing interactions in mature RT. Overall, our data show that the thumb subdomain folds independently and essentially the same in isolation as in its natural structural context.

HIV-1 Capsid Function Is Regulated by Dynamics: Quantitative Atomic-Resolution Insights by Integrating Magic-Angle-Spinning NMR, QM/MM, and MD.

HIV-1 CA capsid protein possesses intrinsic conformational flexibility, which is essential for its assembly into conical capsids and interactions with host factors. CA is dynamic in the assembled capsid, and residues in functionally important regions of the protein undergo motions spanning many decades of time scales. Chemical shift anisotropy (CSA) tensors, recorded in magic-angle-spinning NMR experiments, provide direct residue-specific probes of motions on nano- to microsecond time scales. We combined NMR, MD, and density-functional-theory calculations, to gain quantitative understanding of internal backbone dynamics in CA assemblies, and we found that the dynamically averaged 15N CSA tensors calculated by this joined protocol are in remarkable agreement with experiment. Thus, quantitative atomic-level understanding of the relationships between CSA tensors, local backbone structure, and motions in CA assemblies is achieved, demonstrating the power of integrating NMR experimental data and theory for characterizing atomic-resolution dynamics in biological systems.

Nuclear Magnetic Resonance Structure of the APOBEC3B Catalytic Domain: Structural Basis for Substrate Binding and DNA Deaminase Activity.

Human APOBEC3B (A3B) is a member of the APOBEC3 (A3) family of cytidine deaminases, which function as DNA mutators and restrict viral pathogens and endogenous retrotransposons. Recently, A3B was identified as a major source of genetic heterogeneity in several human cancers. Here, we determined the solution nuclear magnetic resonance structure of the catalytically active C-terminal domain (CTD) of A3B and performed detailed analyses of its deaminase activity. The core of the structure comprises a central five-stranded ß-sheet with six surrounding helices, common to all A3 proteins. The structural fold is most similar to that of A3A and A3G-CTD, with the most prominent difference being found in loop 1. The catalytic activity of A3B-CTD is ∼15-fold lower than that of A3A, although both exhibit a similar pH dependence. Interestingly, A3B-CTD with an A3A loop 1 substitution had significantly increased deaminase activity, while a single-residue change (H29R) in A3A loop 1 reduced A3A activity to the level seen with A3B-CTD. This establishes that loop 1 plays an important role in A3-catalyzed deamination by precisely positioning the deamination-targeted C into the active site. Overall, our data provide important insights into the determinants of the activities of individual A3 proteins and facilitate understanding of their biological function.

Dynamic Nuclear Polarization Enhanced MAS NMR Spectroscopy for Structural Analysis of HIV-1 Protein Assemblies.

Mature infectious HIV-1 virions contain conical capsids composed of CA protein, generated by the proteolytic cleavage cascade of the Gag polyprotein, termed maturation. The mechanism of capsid core formation through the maturation process remains poorly understood. We present DNP-enhanced MAS NMR studies of tubular assemblies of CA and Gag CA-SP1 maturation intermediate and report 20-64-fold sensitivity enhancements due to DNP at 14.1 T. These sensitivity enhancements enabled direct observation of spacer peptide 1 (SP1) resonances in CA-SP1 by dipolar-based correlation experiments, unequivocally indicating that the SP1 peptide is unstructured in assembled CA-SP1 at cryogenic temperatures, corroborating our earlier results. Furthermore, the dependence of DNP enhancements and spectral resolution on magnetic field strength (9.4-18.8 T) and temperature (109-180 K) was investigated. Our results suggest that DNP-based measurements could potentially provide residue-specific dynamics information by allowing for the extraction of the temperature dependence of the anisotropic tensorial or relaxation parameters. With DNP, we were able to detect multiple well-resolved isoleucine side-chain conformers; unique intermolecular correlations across two CA molecules; and functionally relevant conformationally disordered states such as the 14-residue SP1 peptide, none of which are visible at ambient temperatures. The detection of isolated conformers and intermolecular correlations can provide crucial constraints for structure determination of these assemblies. Overall, our results establish DNP-based MAS NMR spectroscopy as an excellent tool for the characterization of HIV-1 assemblies.

Dynamic allostery governs cyclophilin A-HIV capsid interplay.

Host factor protein Cyclophilin A (CypA) regulates HIV-1 viral infectivity through direct interactions with the viral capsid, by an unknown mechanism. CypA can either promote or inhibit viral infection, depending on host cell type and HIV-1 capsid (CA) protein sequence. We have examined the role of conformational dynamics on the nanosecond to millisecond timescale in HIV-1 CA assemblies in the escape from CypA dependence, by magic-angle spinning (MAS) NMR and molecular dynamics (MD). Through the analysis of backbone (1)H-(15)N and (1)H-(13)C dipolar tensors and peak intensities from 3D MAS NMR spectra of wild-type and the A92E and G94D CypA escape mutants, we demonstrate that assembled CA is dynamic, particularly in loop regions. The CypA loop in assembled wild-type CA from two strains exhibits unprecedented mobility on the nanosecond to microsecond timescales, and the experimental NMR dipolar order parameters are in quantitative agreement with those calculated from MD trajectories. Remarkably, the CypA loop dynamics of wild-type CA HXB2 assembly is significantly attenuated upon CypA binding, and the dynamics profiles of the A92E and G94D CypA escape mutants closely resemble that of wild-type CA assembly in complex with CypA. These results suggest that CypA loop dynamics is a determining factor in HIV-1's escape from CypA dependence.

Structural Basis of Clade-specific Engagement of SAMHD1 (Sterile α Motif and Histidine/Aspartate-containing Protein 1) Restriction Factors by Lentiviral Viral Protein X (Vpx) Virulence Factors.

Sterile α motif (SAM) and histidine/aspartate (HD)-containing protein 1 (SAMHD1) restricts human/simian immunodeficiency virus infection in certain cell types and is counteracted by the virulence factor Vpx. Current evidence indicates that Vpx recruits SAMHD1 to the Cullin4-Ring Finger E3 ubiquitin ligase (CRL4) by facilitating an interaction between SAMHD1 and the substrate receptor DDB1- and Cullin4-associated factor 1 (DCAF1), thereby targeting SAMHD1 for proteasome-dependent down-regulation. Host-pathogen co-evolution and positive selection at the interfaces of host-pathogen complexes are associated with sequence divergence and varying functional consequences. Two alternative interaction interfaces are used by SAMHD1 and Vpx: the SAMHD1 N-terminal tail and the adjacent SAM domain or the C-terminal tail proceeding the HD domain are targeted by different Vpx variants in a unique fashion. In contrast, the C-terminal WD40 domain of DCAF1 interfaces similarly with the two above complexes. Comprehensive biochemical and structural biology approaches permitted us to delineate details of clade-specific recognition of SAMHD1 by lentiviral Vpx proteins. We show that not only the SAM domain but also the N-terminal tail engages in the DCAF1-Vpx interaction. Furthermore, we show that changing the single Ser-52 in human SAMHD1 to Phe, the residue found in SAMHD1 of Red-capped monkey and Mandrill, allows it to be recognized by Vpx proteins of simian viruses infecting those primate species, which normally does not target wild type human SAMHD1 for degradation.

Sequence and structural determinants of human APOBEC3H deaminase and anti-HIV-1 activities.

BACKGROUND: Human APOBEC3H (A3H) belongs to the A3 family of host restriction factors, which are cytidine deaminases that catalyze conversion of deoxycytidine to deoxyuridine in single-stranded DNA. A3 proteins contain either one (A3A, A3C, A3H) or two (A3B, A3D, A3F, A3G) Zn-binding domains. A3H has seven haplotypes (I-VII) that exhibit diverse biological phenotypes and geographical distribution in the human population. Its single Zn-coordinating deaminase domain belongs to a phylogenetic cluster (Z3) that is different from the Z1- and Z2-type domains in other human A3 proteins. A3H HapII, unlike A3A or A3C, has potent activity against HIV-1. Here, we sought to identify the determinants of A3H HapII deaminase and antiviral activities, using site-directed sequence- and structure-guided mutagenesis together with cell-based, biochemical, and HIV-1 infectivity assays. RESULTS: We have constructed a homology model of A3H HapII, which is similar to the known structures of other A3 proteins. The model revealed a large cluster of basic residues (not present in A3A or A3C) that are likely to be involved in nucleic acid binding. Indeed, RNase A pretreatment of 293T cell lysates expressing A3H was shown to be required for detection of deaminase activity, indicating that interaction with cellular RNAs inhibits A3H catalytic function. Similar observations have been made with A3G. Analysis of A3H deaminase substrate specificity demonstrated that a 5' T adjacent to the catalytic C is preferred. Changing the putative nucleic acid binding residues identified by the model resulted in reduction or abrogation of enzymatic activity, while substituting Z3-specific residues in A3H to the corresponding residues in other A3 proteins did not affect enzyme function. As shown for A3G and A3F, some A3H mutants were defective in catalysis, but retained antiviral activity against HIV-1vif (-) virions. Furthermore, endogenous reverse transcription assays demonstrated that the E56A catalytic mutant inhibits HIV-1 DNA synthesis, although not as efficiently as wild type. CONCLUSIONS: The molecular and biological activities of A3H are more similar to those of the double-domain A3 proteins than to those of A3A or A3C. Importantly, A3H appears to use both deaminase-dependent and -independent mechanisms to target reverse transcription and restrict HIV-1 replication.

Dissection of binding between a phosphorylated tyrosine hydroxylase peptide and 14-3-3zeta: A complex story elucidated by NMR.

Human tyrosine hydroxylase activity is regulated by phosphorylation of its N-terminus and by an interaction with the modulator 14-3-3 proteins. We investigated the binding of singly or doubly phosphorylated and thiophosphorylated peptides, comprising the first 50 amino acids of human tyrosine hydroxylase, isoform 1 (hTH1), that contain the critical interaction domain, to 14-3-3?, by (31)P NMR. Single phosphorylation at S19 generates a high affinity 14-3-3? binding epitope, whereas singly S40-phosphorylated peptide interacts with 14-3-3? one order-of-magnitude weaker than the S19-phosphorylated peptide. Analysis of the binding data revealed that the 14-3-3? dimer and the S19- and S40-doubly phosphorylated peptide interact in multiple ways, with three major complexes formed: 1), a single peptide bound to a 14-3-3? dimer via the S19 phosphate with the S40 phosphate occupying the other binding site; 2), a single peptide bound to a 14-3-3? dimer via the S19 phosphorous with the S40 free in solution; or 3), a 14-3-3? dimer with two peptides bound via the S19 phosphorous to each binding site. Our system and data provide information as to the possible mechanisms by which 14-3-3 can engage binding partners that possess two phosphorylation sites on flexible tails. Whether these will be realized in any particular interacting pair will naturally depend on the details of each system.

The p66 immature precursor of HIV-1 reverse transcriptase.

In contrast to the wealth of structural data available for the mature p66/p51 heterodimeric human immunodeficiency virus type 1 reverse transcriptase (RT), the structure of the homodimeric p66 precursor remains unknown. In all X-ray structures of mature RT, free or complexed, the processing site in the p66 subunit, for generating the p51 subunit, is sequestered into a ß-strand within the folded ribonuclease H (RNH) domain and is not readily accessible to proteolysis, rendering it difficult to propose a simple and straightforward mechanism of the maturation step. Here, we investigated, by solution NMR, the conformation of the RT p66 homodimer. Our data demonstrate that the RNH and Thumb domains in the p66 homodimer are folded and possess conformations very similar to those in mature RT. This finding suggests that maturation models which invoke a complete or predominantly unfolded RNH domain are unlikely. The present study lays the foundation for further in-depth mechanistic investigations at the atomic level.

Binding of HIV-1 Vpr protein to the human homolog of the yeast DNA repair protein RAD23 (hHR23A) requires its xeroderma pigmentosum complementation group C binding (XPCB) domain as well as the ubiquitin-associated 2 (UBA2) domain.

The human homolog of the yeast DNA repair protein RAD23, hHR23A, has been found previously to interact with the human immunodeficiency virus, type 1 accessory protein Vpr. hHR23A is a modular protein containing an N-terminal ubiquitin-like (UBL) domain and two ubiquitin-associated domains (UBA1 and UBA2) separated by a xeroderma pigmentosum complementation group C binding (XPCB) domain. All domains are connected by flexible linkers. hHR23A binds ubiquitinated proteins and acts as a shuttling factor to the proteasome. Here, we show that hHR23A utilizes both the UBA2 and XPCB domains to form a stable complex with Vpr, linking Vpr directly to cellular DNA repair pathways and their probable exploitation by the virus. Detailed structural mapping of the Vpr contacts on hHR23A, by NMR, revealed substantial contact surfaces on the UBA2 and XPCB domains. In addition, Vpr binding disrupts an intramolecular UBL-UBA2 interaction. We also show that Lys-48-linked di-ubiquitin, when binding to UBA1, does not release the bound Vpr from the hHR23A-Vpr complex. Instead, a ternary hHR23A·Vpr·di-Ub(K48) complex is formed, indicating that Vpr does not necessarily abolish hHR23A-mediated shuttling to the proteasome.