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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 (TPX2^α5-α7) on the microtubule lattice determined by magic-angle-spinning NMR. We demonstrate that TPX2^α5-α7forms 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 TPX2^α5-α7is disordered in solution and adopts a folded structure on MTs, indicating that TPX2^α5-α7undergoes structural changes from unfolded to folded states upon binding to microtubules. The aromatic residues form dense interactions in the core, which stabilize folding of TPX2^α5-α7on microtubules. This work informs on how the phase-separated TPX2^α5-α7behaves on microtubules and represents an atomic-level structural characterization of a protein that is involved in a condensate on cytoskeletal filaments.

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 (CA_CTD), by binding to and stabilizing the CA_CTD-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 CA_CTD-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.

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.