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Initial MAS NMR structure of full-length CA protein Ensemble of 10 lowest-energy structures for the single-chain. Best-fit superpositions for the NTD (a) and CTD (b), respectively, are shown. The color coding is identical to that in Extended Data Fig. 1.
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HIV-1 capsid plays multiple key roles in viral replication, and inhibition of capsid assembly is an attractive target for therapeutic intervention. Here, we report the atomic-resolution structure of capsid protein (CA) tubes, determined by magic-angle spinning NMR and data-guided molecular dynamics simulations. Functionally important regions, inclu...
Citations
... Numerous structural studies first defined the capsid monomer, hexamer, and pentamer building blocks (Mortuza et al., 2004;Pornillos et al., 2009;Pornillos et al., 2011;Gres et al., 2015) ( Figure 1A and B). Subsequent cryo-electron microscopy (cryoEM), molecular dynamics, and solidstate NMR studies of whole retroviral cores and in vitro assemblies have provided insight into capsid assembly and the 12-pentamer fullerene cone geometry (Ganser et al., 1999) of the capsid lattice at high resolution (Zhao et al., 2013;Mattei et al., 2016;Perilla and Schulten, 2017;Lu et al., 2020;Schirra et al., 2023) ( Figure 1C). In the assembly, the capsid N-terminal domains (CA-NTDs) form hexamers and pentamers that are displayed on the outer surface of the shell, while the capsid C-terminal domains (CA-CTDs) form a lower layer that is located at dimer and trimer interfaces to assemble the hexamers and pentamers into the closed fullerene cone. ...
Lenacapavir, targeting the HIV-1 capsid, is the first-in-class antiretroviral drug recently approved for clinical use. The development of Lenacapavir is attributed to the remarkable progress in our understanding of the capsid protein made during the last few years. Considered little more than a component of the virus shell to be shed early during infection, capsid has been found to be a key player in the HIV-1 life cycle by interacting with multiple host cell factors, entering the nucleus, and directing integration. Here, we describe the key advances that led to this “capsid revolution”.
... Owing to deuteration strategies (Akbey et al. 2009;Chevelkov et al. 2006;Linser et al. 2011b;Reif 2021;Vasa et al. 2018a) and facilitated by increasing Magic-Angle-Spinning (MAS) frequencies (Cala-De Paepe et al. 2017;Lewandowski et al. 2011;Schledorn et al. 2020), effectively reducing the influence of coherent contributions from proton-proton dipolar couplings (Böckmann et al. 2015;Malär et al. 2019;Penzel et al. 2019), proton detection has been including various sample types from membrane proteins (Lalli et al. 2017;Retel et al. 2017;Schubeis et al. 2020;Shi et al. 2019;Zhou et al. 2012) and protein amyloids (Becker et al. 2023;Stanek et al. 2016;Xiang et al. 2017) to supramolecular assemblies (Lu et al. 2020;Zinke et al. 2020) and others. Moreover, complex magnetization transfer pathways, correlating multiple nuclei similarly to solution NMR, are becoming increasingly established (Ahlawat et al. 2023;Barbet-Massin et al. 2014;Fraga et al. 2017;Klein et al. 2022a, b;Linser et al. 2010b;Orton et al. 2020;Penzel et al. 2015;Sharma et al. 2020;Stanek et al. 2020;Vasa et al. 2018b). ...
H-detected solid-state NMR spectroscopy has been becoming increasingly popular for the characterization of protein structure, dynamics, and function. Recently, we showed that higher-dimensionality solid-state NMR spectroscopy can aid resonance assignments in large micro-crystalline protein targets to combat ambiguity (Klein et al., Proc. Natl. Acad. Sci. U.S.A. 2022). However, assignments represent both, a time-limiting factor and one of the major practical disadvantages within solid-state NMR studies compared to other structural-biology techniques from a very general perspective. Here, we show that 5D solid-state NMR spectroscopy is not only justified for high-molecular-weight targets but will also be a realistic and practicable method to streamline resonance assignment in small to medium-sized protein targets, which such methodology might not have been expected to be of advantage for. Using a combination of non-uniform sampling and the signal separating algorithm for spectral reconstruction on a deuterated and proton back-exchanged micro-crystalline protein at fast magic-angle spinning, direct amide-to-amide correlations in five dimensions are obtained with competitive sensitivity compatible with common hardware and measurement time commitments. The self-sufficient backbone walks enable efficient assignment with very high confidence and can be combined with higher-dimensionality sidechain-to-backbone correlations from protonated preparations into minimal sets of experiments to be acquired for simultaneous backbone and sidechain assignment. The strategies present themselves as potent alternatives for efficient assignment compared to the traditional assignment approaches in 3D, avoiding user misassignments derived from ambiguity or loss of overview and facilitating automation. This will ease future access to NMR-based characterization for the typical solid-state NMR targets at fast MAS.
... Solid state NMR (ssNMR) is the ideal structural biology technique to characterize insoluble biomolecular aggregates that lack the perfect structural order. [1][2][3][4][5][6][7] The assignment of resonance signals, called chemical shifts (CSs), to specific sites in the molecule, is the pre-requisite to extract site-specific structural information. However, it is challenging to make signal assignments with large proteins (≥150 residues), which seriously cripples the application of ssNMR. ...
Accurate signal assignments can be challenging for congested solid-state NMR (ssNMR) spectra. We describe an automatic sequential assignment program (ASAP) to partially overcome this challenge. ASAP takes three input files: the residue type assignments (RTAs) determined from the better-resolved NCACX spectrum, the full peak list of the NCOCX spectrum, and the protein sequence. It integrates our auto-residue type assignment strategy (ARTIST) with the Monte Carlo simulated annealing (MCSA) algorithm to overcome the hurdle for accurate signal assignments caused by incomplete side-chain resonances and spectral congestion. Combined, ASAP demonstrates robust performance and reduces the time of signal assignments of large proteins (> 200 residues) from years to days.
... [27] Solid-state NMR may overcome these limitations, and it is already used to investigate noncrystalline protein samples, biologics and biomaterials. [28][29][30][31][32][33][34][35][36][37][38][39] Significant enhancements in sensitivity have been obtained by the recent achievements in the NMR probe technology and in biomolecular Dynamic Nuclear Polarization (DNP). [40][41][42][43][44] We here report the design and synthesis of a new molecule that results from the conjugation of the cytotoxic Paclitaxel with Tafamidis (Scheme 1) to form a stable non-covalent protein-drug conjugate (PDC) with TTR. ...
Several protein‐drug conjugates are currently being used in cancer therapy. These conjugates rely on cytotoxic organic compounds that are covalently attached to the carrier proteins or that interact with them via non‐covalent interactions. Human transthyretin (TTR), a physiological protein, has already been identified as a possible carrier protein for the delivery of cytotoxic drugs. Here we show the structure‐guided development of a new stable cytotoxic molecule based on a known strong binder of TTR and a well‐established anticancer drug. This example is used to demonstrate the importance of the integration of multiple biophysical and structural techniques, encompassing microscale thermophoresis, X‐ray crystallography and NMR. In particular, we show that solid‐state NMR has the ability to reveal effects caused by ligand binding which are more easily relatable to structural and dynamical alterations that impact the stability of macromolecular complexes.
... Bounds of the distance restraints were set to 1.5-6.5 Å (4.0 ± 2.5 Å) and 2.0-7.2 Å (4.6 ± 2.6 Å) for intra-and inter-residue restraints, respectively, consistent with our previous studies [85][86][87] . Dihedral restraints were predicted using TALOS-N 88 from the experimental solid-state 1 H, 13 C and 15 N chemical shifts. ...
Targeting protein for Xklp2 (TPX2) is a key factor that stimulates branching microtubule nucleation during cell division. Upon binding to microtubules (MTs), TPX2 forms condensates via liquid-liquid phase separation, which facilitates recruitment of microtubule nucleation factors and tubulin. We report the structure of the TPX2 C-terminal minimal active domain (TPX2α5-α7) on the microtubule lattice determined by magic-angle-spinning NMR. We demonstrate that TPX2α5-α7 forms a co-condensate with soluble tubulin on microtubules and binds to MTs between two adjacent protofilaments and at the intersection of four tubulin heterodimers. These interactions stabilize the microtubules and promote the recruitment of tubulin. Our results reveal that TPX2α5-α7 is disordered in solution and adopts a folded structure on MTs, indicating that TPX2α5-α7 undergoes structural changes from unfolded to folded states upon binding to microtubules. The aromatic residues form dense interactions in the core, which stabilize folding of TPX2α5-α7 on microtubules. This work informs on how the phase-separated TPX2α5-α7 behaves on microtubules and represents an atomic-level structural characterization of a protein that is involved in a condensate on cytoskeletal filaments.
... Modulation of HIV-1 IP5/6 dependence by the capsid lattice Fig 1F was modeled in UCSF Chimera by aligning the NMR structure of the full-length HIV-1 CA protein (PDB 6WAP, [76]) to a portion of the cryo-electron microscopy structure of the HIV-1 capsid (PDB 5MDG, [77]). Figs 2A, 2B, S1A and S1B show an atomic model of the HIV-1 capsid (PDB 6J3Y, [42]) with the indicated residues mapped onto it using UCSF Chimera. ...
The mature HIV-1 capsid is stabilized by host and viral determinants. The capsid protein CA binds to the cellular metabolites inositol hexakisphosphate (IP6) and its precursor inositol (1, 3, 4, 5, 6) pentakisphosphate (IP5) to stabilize the mature capsid. In target cells, capsid destabilization by the antiviral compounds lenacapavir and PF74 reveals a HIV-1 infectivity defect due to IP5/IP6 (IP5/6) depletion. To test whether intrinsic HIV-1 capsid stability and/or host factor binding determines HIV-1 insensitivity to IP5/6 depletion, a panel of CA mutants was assayed for infection of IP5/6-depleted T cells and wildtype cells. Four CA mutants with unstable capsids exhibited dependence on host IP5/6 for infection and reverse transcription (RTN). Adaptation of one such mutant, Q219A, by spread in culture resulted in Vpu truncation and a capsid three-fold interface mutation, T200I. T200I increased intrinsic capsid stability as determined by in vitro uncoating of purified cores and partially reversed the IP5/6-dependence in target cells for each of the four CA mutants. T200I further rescued the changes to lenacapavir sensitivity associated with the parental mutation. The premature dissolution of the capsid caused by the IP5/6-dependent mutations imparted a unique defect in integration targeting that was rescued by T200I. Collectively, these results demonstrate that T200I restored other capsid functions after RTN for the panel of mutants. Thus, the hyperstable T200I mutation stabilized the instability defects imparted by the parental IP5/6-dependent CA mutation. The contribution of Vpu truncation to mutant adaptation was linked to BST-2 antagonization, suggesting that cell-to-cell transfer promoted replication of the mutants. We conclude that interactions at the three-fold interface are adaptable, key mediators of capsid stability in target cells and are able to antagonize even severe capsid instability to promote infection.
... In order to generate a list of distance restraints, home-written Python scripts were provided with the two peak lists and the assigned chemical shifts of ssDNA-bound gVp as input. A chemical shift tolerance window of 0.3 ppm (a value typically chosen for ssNMR spectra 41,54 ) was used in order to attribute all possible assignments to each cross-peak, resulting in a set of both ambiguous distance restraints (ADRs 55 ) and unambiguous restraints (a single possible assignment). All restraints were set to a range of 2−8 Å. 40,56 For restraints arising from the sparsely labeled data sets, we utilized a probability threshold of 40% in order to discard possible assignments, which corresponded to pairs of carbon sites that had a low probability of both being isotopically labeled. ...
F-specific filamentous phages, elongated particles with circular single-stranded DNA encased in a symmetric protein capsid, undergo an intermediate step, where thousands of homodimers of a non-structural protein, gVp, bind to newly synthesized strands of DNA, preventing further DNA replication and preparing the circular genome in an elongated conformation for assembly of a new virion structure at the membrane. While the structure of the free homodimer is known, the ssDNA-bound conformation has yet to be determined. We report an atomic-resolution structure of the gVp monomer bound to ssDNA of fd phage in the nucleoprotein complex elucidated via magic-angle spinning solid-state NMR. The model presents significant conformational changes with respect to the free form. These modifications facilitate the binding mechanism and possibly promote cooperative binding in the assembly of the gVp–ssDNA complex.
... Magic-anglespinning (MAS) NMR is a powerful technique for atomic-resolution structure determinations of proteins in biological assemblies and yields residue-specific structural and dynamics information. Previous studies have demonstrated that accurate and precise protein structures can be determined by integrating NMR experimental distance restraints with low-to-medium resolution cryo-EM density maps for large biomolecular complexes [36][37][38][39][40] . Using this integrated approach, fewer restraints are required and the structures can be refined to higher precision and accuracy when cryo-EM maps are available. ...
... Bounds of the distance restraints were set to 1.5-6.5 Å (4.0 ± 2.5 Å) and 2.0-7.2 Å (4.6 ± 2.6 Å) for intra-and inter-residue restraints, respectively, consistent with our previous study 39 . ...
... The lowest energy structure from the run described in the last subsection was subjected to rigid-body docking about the experimental cryo-EM density map (EMD-6187, PDB 3J8X, 6 Å resolution) using an in-house UCSF Chimera script. The protocol bears similarities to previous work from our laboratory 39 with an important adaptation: instead of employing docking to identify the best fitting structure amongst many candidates in the cryo-EM density, the script identifies the best docking positions of a single structure. Here, 22 positions were identified about the map on the basis of the lowest cross-correlation values and brief visual inspection. ...
Microtubules (MTs) and their associated proteins play essential roles in maintaining cell structure, organelle transport, cell motility, and cell division. Two motors, kinesin and cytoplasmic dynein link the MT network to transported cargos using ATP for force generation. Here, we report an all-atom NMR structure of nucleotide-free kinesin-1 motor domain (apo-KIF5B) in complex with paclitaxel-stabilized microtubules using magic-angle-spinning (MAS) NMR spectroscopy. The structure reveals the position and orientation of the functionally important neck linker and how ADP induces structural and dynamic changes that ensue in the neck linker. These results demonstrate that the neck linker is in the undocked conformation and oriented in the direction opposite to the KIF5B movement. Chemical shift perturbations and intensity changes indicate that a significant portion of ADP-KIF5B is in the neck linker docked state. This study also highlights the unique capability of MAS NMR to provide atomic-level information on dynamic regions of biological assemblies.
... Exploiting these capabilities, SSNMR has been used to elucidate structures of complex assemblies similar in size to those studied by cryo-EM while in their native state, without the need for cryogenic preservation. As of mid-2022, the largest macromolecular structure determined by MAS SSNMR is the HIV-1 Capsid Tube, containing 378 repeats of a 231-residue subunit for a total of 87,318 residues (PDB ID 6x63 [92]). Larger structures have also been determined using integrative or hybrid methods, including that of a 484.61 kDa, 24mer B-crystallin oligomer (4200 residues), incorporating experimental data from solution NMR, solution scattering, and 3DEM (PDB ID 3j07 [93]), and that of the 470.42 kDa tetrahedral aminopeptidase TET2 (4236 residues total), incorporating data from SSNMR and 3DEM (PDB ID 6r8n [94]). ...
The Research Collaboratory for Structural Bioinformatics Protein Data Bank (RCSB PDB), funded by the United States National Science Foundation, National Institutes of Health, and Department of Energy, supports structural biologists and Protein Data Bank (PDB) data users around the world. The RCSB PDB, a founding member of the Worldwide Protein Data Bank (wwPDB) partnership, serves as the US data center for the global PDB archive housing experimentally-determined three-dimensional (3D) structure data for biological macromolecules. As the wwPDB-designated Archive Keeper, RCSB PDB is also responsible for the security of PDB data and weekly update of the archive. RCSB PDB serves tens of thousands of data depositors (using macromolecular crystallography, nuclear magnetic resonance spectroscopy, electron microscopy, and micro-electron diffraction) annually working on all permanently inhabited continents. RCSB PDB makes PDB data available from its research-focused RCSB.org web portal at no charge and without usage restrictions to many millions of PDB data consumers around the globe. It also provides educators, students, and the general public with an introduction to the PDB and related training materials through its outreach and education-focused web portal, PDB101.RCSB.org. This review article describes growth of the PDB, examines evolution of experimental methods for structure determination viewed through the lens of the PDB archive, and provides a detailed accounting of PDB archival holdings and their utilization by researchers, educators, and students worldwide.
... We set out to determine how the C-terminal region of TPX2, which contains the domains a5-a7 that are essential for its activity ( Figure 1A, amino acids 477-716), binds to MTs, a prerequisite to elicit branching MT nucleation 29,30 . As shown in Figure 1B-C, GFP-tagged TPX2 a5-a7 co-localizes with branched microtubule networks in Xenopus egg extract and binds to pre-formed GMPCPP-stabilized microtubules in vitro, consistent with previous results. ...
... Bounds of the distance restraints were set to 1.5-6.5 Å (4.0 ± 2.5 Å) and 2.0-7.2 Å (4.6 ± 2.6 Å) for intra-and inter-residue restraints, respectively, consistent with our previous studies 30,31 . Dihedral restraints were predicted using TALOS-N 32 from the experimental solid-state 13 C and 15 N chemical shifts. ...
Targeting protein for Xklp2 (TPX2) is a key factor that stimulates branching microtubule nucleation during cell division. Upon binding to microtubules, TPX2 forms condensates via liquid-liquid phase separation, which facilitates recruitment of microtubule nucleation factors and tubulin. We report the structure of the TPX2 C-terminal minimal active domain (TPX2 α5-α7 ) on the microtubule lattice, determined by magic-angle-spinning NMR. We demonstrate that TPX2 α5-α7 can form a co-condensate with soluble tubulin and a liquid layer on microtubules, by binding between two adjacent protofilaments and at the intersection of four tubulin heterodimers. These interactions stabilize the microtubules and promote the recruitment of tubulin. Aromatic residues form dense hydrophobic interactions that stabilize folding of TPX2 α5-α7 . This work informs on how the phase-separated TPX2 α5-α7 behaves on microtubules and represents the first structural characterization of a protein that forms a liquid layer on a cytoskeletal filament.
