Implications for kinetochore-microtubule attachment from the structure of an engineered Ndc80 complex.

Kinetochores are proteinaceous assemblies that mediate the interaction of chromosomes with the mitotic spindle. The 180 kDa Ndc80 complex is a direct point of contact between kinetochores and microtubules. Its four subunits contain coiled coils and form an elongated rod structure with functional globular domains at either end. We crystallized an engineered "bonsai" Ndc80 complex containing a shortened rod domain but retaining the globular domains required for kinetochore localization and microtubule binding. The structure reveals a microtubule-binding interface containing a pair of tightly interacting calponin-homology (CH) domains with a previously unknown arrangement. The interaction with microtubules is cooperative and predominantly electrostatic. It involves positive charges in the CH domains and in the N-terminal tail of the Ndc80 subunit and negative charges in tubulin C-terminal tails and is regulated by the Aurora B kinase. We discuss our results with reference to current models of kinetochore-microtubule attachment and centromere organization.

Structure of a Na+/H+ antiporter and insights into mechanism of action and regulation by pH.

The control by Na+/H+ antiporters of sodium/proton concentration and cell volume is crucial for the viability of all cells. Adaptation to high salinity and/or extreme pH in plants and bacteria or in human heart muscles requires the action of Na+/H+ antiporters. Their activity is tightly controlled by pH. Here we present the crystal structure of pH-downregulated NhaA, the main antiporter of Escherichia coli and many enterobacteria. A negatively charged ion funnel opens to the cytoplasm and ends in the middle of the membrane at the putative ion-binding site. There, a unique assembly of two pairs of short helices connected by crossed, extended chains creates a balanced electrostatic environment. We propose that the binding of charged substrates causes an electric imbalance, inducing movements, that permit a rapid alternating-access mechanism. This ion-exchange machinery is regulated by a conformational change elicited by a pH signal perceived at the entry to the cytoplasmic funnel.

Crucial steps in the structure determination of the Na+/H+ antiporter NhaA in its native conformation.

Sodium proton antiporters are ubiquitous membrane proteins. Their importance for cell viability is the result of their role in homeostasis of intracellular pH, cellular Na+ content and cell volume. Recently, the first structure of this family of secondary transporters, namely of NhaA from Escherichia coli, revealed a novel fold and elucidated the molecular basis for the mechanism of transport and its regulation by pH. Here, we describe the key steps for the structure determination of NhaA, an iterative process of improving protein quality as well as crystallization conditions. Protein quality was optimized by shortening the purification to a single step and by changing the expression host. The major steps for crystal improvement were the exchange of the detergent during protein purification from the beta- to the alpha-anomer of DDM, the addition of OG to the crystallization set ups, and the growth of the crystals under conditions suitable for cryo-temperatures. Unexpectedly, the dimeric association of the transporter in the 3D crystal lattice is non-physiological. A comparison of the X-ray structure with the electron density map from cryo-electron microscopy of 2D crystals demonstrates that the NhaA helix packing in the 3D crystal is identical with the one in the lipid environment. Thus, the antiporter is in a native conformation in the 3D crystals.

Direct binding of Cenp-C to the Mis12 complex joins the inner and outer kinetochore.

Kinetochores are proteinaceous scaffolds implicated in the formation of load-bearing attachments of chromosomes to microtubules during mitosis. Kinetochores contain distinct chromatin- and microtubule-binding interfaces, generally defined as the inner and outer kinetochore, respectively (reviewed in). The constitutive centromere-associated network (CCAN) and the Knl1-Mis12-Ndc80 complexes (KMN) network are the main multisubunit protein assemblies in the inner and outer kinetochore, respectively. The point of contact between the CCAN and the KMN network is unknown. Cenp-C is a conserved CCAN component whose central and C-terminal regions have been implicated in chromatin binding and dimerization. Here, we show that a conserved motif in the N-terminal region of Cenp-C binds directly and with high affinity to the Mis12 complex. Expression in HeLa cells of the isolated N-terminal motif of Cenp-C prevents outer kinetochore assembly, causing chromosome missegregation. The KMN network is also responsible for kinetochore recruitment of the components of the spindle assembly checkpoint, and we observe checkpoint impairment in cells expressing the Cenp-C N-terminal segment. Our studies unveil a crucial and likely universal link between the inner and outer kinetochore.

A screen for kinetochore-microtubule interaction inhibitors identifies novel antitubulin compounds.

BACKGROUND: Protein assemblies named kinetochores bind sister chromatids to the mitotic spindle and orchestrate sister chromatid segregation. Interference with kinetochore activity triggers a spindle checkpoint mediated arrest in mitosis, which frequently ends in cell death. We set out to identify small compounds that inhibit kinetochore-microtubule binding for use in kinetochore-spindle interaction studies and to develop them into novel anticancer drugs. METHODOLOGY/PRINCIPAL FINDINGS: A fluorescence microscopy-based in vitro assay was developed to screen compound libraries for molecules that prevented the binding of a recombinant human Ndc80 kinetochore complex to taxol-stabilized microtubules. An active compound was identified that acted at the microtubule level. More specifically, by localizing to the colchicine-binding site in alphabeta-tubulin the hit compound prevented the Ndc80 complex from binding to the microtubule surface. Next, structure-activity analyses distinguished active regions in the compound and led to the identification of highly potent analogs that killed cancer cells with an efficacy equaling that of established spindle drugs. CONCLUSIONS/SIGNIFICANCE: The compound identified in our screen and its subsequently identified analogs represent new antitubulin chemotypes that can be synthetically developed into a novel class of antimitotic spindle drugs. In addition, they are stereochemically unique as their R- and S-isomers mimic binding of colchicine and podophyllotoxin, respectively, two antitubulin drugs that interact differently with the tubulin interface. Model-driven manipulation of our compounds promises to advance insight into how antitubulin drugs act upon tubulin. These advances in turn may lead to tailor-made colchicine site agents which would be valuable new assets to fight a variety of tumors, including those that have become resistant to the (antispindle) drugs used today.

Discontinuous membrane helices in transport proteins and their correlation with function.

Alpha-helical bundles and beta-barrel proteins represent the two basic types of architecture known for integral membrane proteins. Irregular structural motifs have been revealed with the growing number of structures determined. "Discontinuous" helices are present in membrane proteins that actively transport ions. In the Ca(2+)-ATPase, a primary active transporter, and in the secondary transporters NhaA, LeuT(Aa), ClC H(+)/Cl(-) exchanger and Glt(Ph), the helical structure of two membrane segments is interrupted and the interjacent polypeptide chain forms an extended peptide. The discontinuous helices are integrated in the membrane either as transmembrane-spanning or hairpin-type segments. In addition, the secondary transporters have inverted internal duplication domains, which are only weakly correlated with their amino acid sequence. The symmetry comprises either parts of or the complete molecule, but always includes the discontinuous helices. The helix-peptide-helix motif is correlated with the ion translocation function. The extended peptides with their backbone atoms, the helix termini and the polar/charged amino acid residues in close vicinity provide the basis for ion recognition, binding and translocation.

Multiconformation continuum electrostatics analysis of the NhaA Na+/H+ antiporter of Escherichia coli with functional implications.

Sodium proton antiporters are essential enzymes that catalyze the exchange of sodium ions for protons across biological membranes. Protonations and deprotonations of individual amino acid residues and of clusters formed by these residues play an important role in activating these enzymes and in the mechanism of transport. We have used multiconformation continuum electrostatics method to investigate the protonation states of residues in the sodium proton exchanger NhaA from Escherichia coli, the structure of which has been determined recently by x-ray crystallography. Our calculations identify four clusters of electrostatically tightly interacting residues as well as long-range interactions between residues required for activation. The importance of many of these residues has been demonstrated by the characterization of site-directed mutants. A number of residues with extreme pKa values, including several of the "pH sensor," can only undergo protonation/deprotonation reactions subsequent to conformational changes. The results of the calculations provide valuable information on the activation of the antiporter and the role of individual amino acid residues, and provide a solid framework for further experiments.