A communication hub for phosphoregulation of kinetochore-microtubule attachment.

The Mps1 and Aurora B kinases regulate and monitor kinetochore attachment to spindle microtubules during cell division, ultimately ensuring accurate chromosome segregation. In yeast, the critical spindle attachment components are the Ndc80 and Dam1 complexes (Ndc80c and DASH/Dam1c, respectively). Ndc80c is a 600-Å-long heterotetramer that binds microtubules through a globular "head" at one end and centromere-proximal kinetochore components through a globular knob at the other end. Dam1c is a heterodecamer that forms a ring of 16-17 protomers around the shaft of the single kinetochore microtubule in point-centromere yeast. The ring coordinates the approximately eight Ndc80c rods per kinetochore. In published work, we showed that a site on the globular "head" of Ndc80c, including residues from both Ndc80 and Nuf2, binds a bipartite segment in the long C-terminal extension of Dam1. Results reported here show, both by in vitro binding experiments and by crystal structure determination, that the same site binds a conserved segment in the long N-terminal extension of Mps1. It also binds, less tightly, a conserved segment in the N-terminal extension of Ipl1 (yeast Aurora B). Together with results from experiments in yeast cells and from biochemical assays reported in two accompanying papers, the structures and graded affinities identify a communication hub for ensuring uniform bipolar attachment and for signaling anaphase onset.

Structure of the Ndc80 complex and its interactions at the yeast kinetochore-microtubule interface.

The conserved Ndc80 kinetochore complex, Ndc80c, is the principal link between mitotic spindle microtubules and centromere-associated proteins. We used AlphaFold 2 (AF2) to obtain predictions of the Ndc80 'loop' structure and of the Ndc80 : Nuf2 globular head domains that interact with the Dam1 subunit of the heterodecameric DASH/Dam1 complex (Dam1c). The predictions guided design of crystallizable constructs, with structures close to the predicted ones. The Ndc80 'loop' is a stiff, α-helical 'switchback' structure; AF2 predictions and positions of preferential cleavage sites indicate that flexibility within the long Ndc80c rod occurs instead at a hinge closer to the globular head. Conserved stretches of the Dam1 C terminus bind Ndc80c such that phosphorylation of Dam1 serine residues 257, 265 and 292 by the mitotic kinase Ipl1/Aurora B can release this contact during error correction of mis-attached kinetochores. We integrate the structural results presented here into our current molecular model of the kinetochore-microtubule interface. The model illustrates how multiple interactions between Ndc80c, DASH/Dam1c and the microtubule lattice stabilize kinetochore attachments.

Bound nucleotide can control the dynamic architecture of monomeric actin.

Polymerization of actin into cytoskeletal filaments is coupled to its bound adenine nucleotides. The mechanism by which nucleotide modulates actin functions has not been evident from analyses of ATP- and ADP-bound crystal structures of the actin monomer. We report that NMR chemical shift differences between the two forms are globally distributed. Furthermore, microsecond-millisecond motions are spread throughout the molecule in the ATP form, but largely confined to subdomains 1 and 2, and the nucleotide binding site in the ADP form. Through these motions, the ATP- and ADP-bound forms sample different high-energy conformations. A deafness-causing, fast-nucleating actin mutant populates the high-energy conformer of ATP-actin more than the wild-type protein, suggesting that this conformer may be on the pathway to nucleation. Together, the data suggest a model in which differential sampling of a nucleation-compatible form of the actin monomer may contribute to control of actin filament dynamics by nucleotide.

Structural basis of Stu2 recruitment to yeast kinetochores.

Chromosome segregation during cell division requires engagement of kinetochores of sister chromatids with microtubules emanating from opposite poles. As the corresponding microtubules shorten, these 'bioriented' sister kinetochores experience tension-dependent stabilization of microtubule attachments. The yeast XMAP215 family member and microtubule polymerase, Stu2, associates with kinetochores and contributes to tension-dependent stabilization in vitro. We show here that a C-terminal segment of Stu2 binds the four-way junction of the Ndc80 complex (Ndc80c) and that residues conserved both in yeast Stu2 orthologs and in their metazoan counterparts make specific contacts with Ndc80 and Spc24. Mutations that perturb this interaction prevent association of Stu2 with kinetochores, impair cell viability, produce biorientation defects, and delay cell cycle progression. Ectopic tethering of the mutant Stu2 species to the Ndc80c junction restores wild-type function in vivo. These findings show that the role of Stu2 in tension-sensing depends on its association with kinetochores by binding with Ndc80c.

Improved strategy for isoleucine 1H/13C methyl labeling in Pichia pastoris.

Site specific methyl labeling combined with methyl TROSY offers a powerful NMR approach to study structure and dynamics of proteins and protein complexes of high molecular weight. Robust and cost-effective methods have been developed for site specific protein 1H/13C methyl labeling in an otherwise deuterated background in bacteria. However, bacterial systems are not suitable for expression and isotope labeling of many eukaryotic and membrane proteins. The yeast Pichia pastoris (P. pastoris) is a commonly used host for expression of eukaryotic proteins, and site-specific methyl labeling of perdeuterated eukaryotic proteins has recently been achieved with this system. However, the practical utility of methyl labeling and deuteration in P. pastoris is limited by high costs. Here, we describe an improved method for 1H/13C-labeling of the δ-methyl group of isoleucine residues in a perdeuterated background, which reduces the cost by ≥ 50% without compromising the efficiency of isotope enrichment. We have successfully implemented this method to label actin and a G-protein coupled receptor. Our approach will facilitate studies of the structure and dynamics of eukaryotic proteins by NMR spectroscopy.

Methyl labeling and TROSY NMR spectroscopy of proteins expressed in the eukaryote Pichia pastoris.

(13)C Methyl TROSY NMR spectroscopy has emerged as a powerful method for studying the dynamics of large systems such as macromolecular assemblies and membrane proteins. Specific (13)C labeling of aliphatic methyl groups and perdeuteration has been limited primarily to proteins expressed in E. coli, preventing studies of many eukaryotic proteins of physiological and biomedical significance. We demonstrate the feasibility of efficient (13)C isoleucine δ1-methyl labeling in a deuterated background in an established eukaryotic expression host, Pichia pastoris, and show that this method can be used to label the eukaryotic protein actin, which cannot be expressed in bacteria. This approach will enable NMR studies of previously intractable targets.

The bacterial effector VopL organizes actin into filament-like structures.

VopL is an effector protein from Vibrio parahaemolyticus that nucleates actin filaments. VopL consists of a VopL C-terminal domain (VCD) and an array of three WASP homology 2 (WH2) motifs. Here, we report the crystal structure of the VCD dimer bound to actin. The VCD organizes three actin monomers in a spatial arrangement close to that found in the canonical actin filament. In this arrangement, WH2 motifs can be modeled into the binding site of each actin without steric clashes. The data suggest a mechanism of nucleation wherein VopL creates filament-like structures, organized by the VCD with monomers delivered by the WH2 array, that can template addition of new subunits. Similarities with Arp2/3 complex and formin proteins suggest that organization of monomers into filament-like structures is a general and central feature of actin nucleation.

Dimerization of Plasmodium vivax DBP is induced upon receptor binding and drives recognition of DARC.

Plasmodium vivax and Plasmodium knowlesi invasion depends on the parasite Duffy-binding protein DBL domain (RII-PvDBP or RII-PkDBP) engaging the Duffy antigen receptor for chemokines (DARC) on red blood cells. Inhibition of this key interaction provides an excellent opportunity for parasite control. There are competing models for whether Plasmodium ligands engage receptors as monomers or dimers, a question whose resolution has profound implications for parasite biology and control. We report crystallographic, solution and functional studies of RII-PvDBP showing that dimerization is required for and driven by receptor engagement. This work provides a unifying framework for prior studies and accounts for the action of naturally acquired blocking antibodies and the mechanism of immune evasion. We show that dimerization is conserved in DBL-domain receptor engagement and propose that receptor-mediated ligand dimerization drives receptor affinity and specificity. Because dimerization is prevalent in signaling, our studies raise the possibility that induced dimerization may activate pathways for invasion.

Mechanisms of human immunodeficiency virus type 1 concerted integration related to strand transfer inhibition and drug resistance.

The "strand transfer inhibitors" of human immunodeficiency virus type-1 (HIV-1) integrase (IN), so named because of their pronounced selectivity for inhibiting strand transfer over 3' OH processing, block virus replication in vivo and ex vivo and prevent concerted integration in vitro. We explored the kinetics of product formation and strand transfer inhibition within reconstituted synaptic complexes capable of concerted integration. Synaptic complexes were formed with viral DNA donors containing either two blunt ends, two 3'-OH-processed ends, or one of each. We determined that one blunt end within a synaptic complex is a sufficient condition for low-nanomolar-range strand transfer inhibition with naphthyridine carboxamide inhibitors L-870,810 and L-870,812. We further explored the catalytic properties and drug resistance profiles of a set of clinically relevant strand transfer inhibitor-resistant HIV-1 IN mutants. The diketo acids and naphthyridine carboxamides, mechanistically similar but structurally distinct strand transfer inhibitors, each select for a distinct set of drug resistance mutations ex vivo. The S153Y and N155S IN resistance mutants were selected with the diketo acid L-841,411, and the N155H mutant was selected with L-810,812. Each mutant exhibited some degree of catalytic impairment relative to the activity of wild type IN, although the N155H mutant displayed near-wild-type IN activities. The resistance profiles indicated that the S153Y mutation potentiates susceptibility to L-870,810 and L-870,812, while the N155S mutation confers resistance to L-870,810 and L-870,812. The N155H mutation confers resistance to L-870,810 and potentiates susceptibility to L-841,411. This study illuminates the interrelated mechanisms of concerted integration, strand transfer inhibition, and resistance to strand transfer inhibitors.