Structural basis of protein condensation on microtubules underlying branching microtubule nucleation.

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.

Structural basis of HIV-1 maturation inhibitor binding and activity.

HIV-1 maturation inhibitors (MIs), Bevirimat (BVM) and its analogs interfere with the catalytic cleavage of spacer peptide 1 (SP1) from the capsid protein C-terminal domain (CACTD), by binding to and stabilizing the CACTD-SP1 region. MIs are under development as alternative drugs to augment current antiretroviral therapies. Although promising, their mechanism of action and associated virus resistance pathways remain poorly understood at the molecular, biochemical, and structural levels. We report atomic-resolution magic-angle-spinning NMR structures of microcrystalline assemblies of CACTD-SP1 complexed with BVM and/or the assembly cofactor inositol hexakisphosphate (IP6). Our results reveal a mechanism by which BVM disrupts maturation, tightening the 6-helix bundle pore and quenching the motions of SP1 and the simultaneously bound IP6. In addition, BVM-resistant SP1-A1V and SP1-V7A variants exhibit distinct conformational and binding characteristics. Taken together, our study provides a structural explanation for BVM resistance as well as guidance for the design of new MIs.

Magic-angle-spinning NMR structure of the kinesin-1 motor domain assembled with microtubules reveals the elusive neck linker orientation.

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.

Atomic-Resolution Structure of SARS-CoV-2 Nucleocapsid Protein N-Terminal Domain.

The nucleocapsid (N) protein is one of the four structural proteins of the SARS-CoV-2 virus and plays a crucial role in viral genome organization and, hence, replication and pathogenicity. The N-terminal domain (NNTD) binds to the genomic RNA and thus comprises a potential target for inhibitor and vaccine development. We determined the atomic-resolution structure of crystalline NNTD by integrating solid-state magic angle spinning (MAS) NMR and X-ray diffraction. Our combined approach provides atomic details of protein packing interfaces as well as information about flexible regions as the N- and C-termini and the functionally important RNA binding, ß-hairpin loop. In addition, ultrafast (100 kHz) MAS 1H-detected experiments permitted the assignment of side-chain proton chemical shifts not available by other means. The present structure offers guidance for designing therapeutic interventions against the SARS-CoV-2 infection.

Magic angle spinning NMR structure of human cofilin-2 assembled on actin filaments reveals isoform-specific conformation and binding mode.

Actin polymerization dynamics regulated by actin-binding proteins are essential for various cellular functions. The cofilin family of proteins are potent regulators of actin severing and filament disassembly. The structural basis for cofilin-isoform-specific severing activity is poorly understood as their high-resolution structures in complex with filamentous actin (F-actin) are lacking. Here, we present the atomic-resolution structure of the muscle-tissue-specific isoform, cofilin-2 (CFL2), assembled on ADP-F-actin, determined by magic-angle-spinning (MAS) NMR spectroscopy and data-guided molecular dynamics (MD) simulations. We observe an isoform-specific conformation for CFL2. This conformation is the result of a unique network of hydrogen bonding interactions within the α2 helix containing the non-conserved residue, Q26. Our results indicate F-site interactions that are specific between CFL2 and ADP-F-actin, revealing mechanistic insights into isoform-dependent F-actin disassembly.

Atomic-resolution structure of HIV-1 capsid tubes by magic-angle spinning NMR.

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, including the NTD ß-hairpin, the cyclophilin A-binding loop, residues in the hexamer central pore, and the NTD-CTD linker region, are well defined. The structure of individual CA chains, their arrangement in the pseudo-hexameric units of the tube and the inter-hexamer interfaces are consistent with those in intact capsids and substantially different from the organization in crystal structures, which feature flat hexamers. The inherent curvature in the CA tubes is controlled by conformational variability of residues in the linker region and of dimer and trimer interfaces. The present structure reveals atomic-level detail in capsid architecture and provides important guidance for the design of novel capsid inhibitors.

Accuracy and precision of protein structures determined by magic angle spinning NMR spectroscopy: for some 'with a little help from a friend'.

We present a systematic investigation into the attainable accuracy and precision of protein structures determined by heteronuclear magic angle spinning solid-state NMR for a set of four proteins of varied size and secondary structure content. Structures were calculated using synthetically generated random sets of C-C distances up to 7 Å at different degrees of completeness. For single-domain proteins, 9-15 restraints per residue are sufficient to derive an accurate model structure, while maximum accuracy and precision are reached with over 15 restraints per residue. For multi-domain proteins and protein assemblies, additional information on domain orientations, quaternary structure and/or protein shape is needed. As demonstrated for the HIV-1 capsid protein assembly, this can be accomplished by integrating MAS NMR with cryoEM data. In all cases, inclusion of TALOS-derived backbone torsion angles improves the accuracy for small number of restraints, while no further increases are noted for restraint completeness above 40%. In contrast, inclusion of TALOS-derived torsion angle restraints consistently increases the precision of the structural ensemble at all degrees of distance restraint completeness.