Faculty Bibliography

Bifurcated electron transfer during ubiquinol oxidation is the key reaction of cytochrome bc1 complex catalysis. Binding of the competitive inhibitor 5-n-heptyl-6-hydroxy-4,7-dioxobenzothiazole to the Qo site of the cytochrome bc1 complex from Saccharomyces cerevisiae was analyzed by x-ray crystallography. This alkylhydroxydioxobenzothiazole is bound in its ionized form as evident from the crystal structure and confirmed by spectroscopic analysis, consistent with a measured pKa = 6.1 of the hydroxy group in detergent micelles. Stabilizing forces for the hydroxyquinone anion inhibitor include a polarized hydrogen bond to the iron-sulfur cluster ligand His181 and on-edge interactions via weak hydrogen bonds with cytochrome b residue Tyr279. The hydroxy group of the latter contributes to stabilization of the Rieske protein in the b-position by donating a hydrogen bond. The reported pH dependence of inhibition with lower efficacy at alkaline pH is attributed to the protonation state of His181 with a pKa of 7.5. Glu272, a proposed primary ligand and proton acceptor of ubiquinol, is not bound to the carbonyl group of the hydroxydioxobenzothiazole ring but is rotated out of the binding pocket toward the heme bL propionate A, to which it is hydrogen-bonded via a single water molecule. The observed hydrogen bonding pattern provides experimental evidence for the previously proposed proton exit pathway involving the heme propionate and a chain of water molecules. Binding of the alkyl-6-hydroxy-4,7-dioxobenzothiazole is discussed as resembling an intermediate step of ubiquinol oxidation, supporting a single occupancy model at the Qo site.

Atovaquone is a substituted 2-hydroxynaphthoquinone that is used therapeutically to treat Plasmodium falciparum malaria, Pneumocystis carinii pneumonia, and Toxoplasma gondii toxoplasmosis. It is thought to act on these organisms by inhibiting the cytochrome bc1 complex. We have examined the interaction of atovaquone with the bc1 complex isolated from Saccharomyces cerevisiae, a surrogate, nonpathogenic fungus. Atovaquone inhibits the bc1 complex competitively with apparent Ki = 9 nm, raises the midpoint potential of the Rieske iron-sulfur protein from 285 to 385 mV, and shifts the g values in the EPR spectrum of the Rieske center. These results indicate that atovaquone binds to the ubiquinol oxidation pocket of the bc1 complex, where it interacts with the Rieske iron-sulfur protein. A computed energy-minimized structure for atovaquone liganded to the yeast bc1 complex suggests that a phenylalanine at position 275 of cytochrome b in the bovine bc1 complex, as opposed to leucine at the equivalent position in the yeast enzyme, is responsible for the decreased sensitivity of the bovine bc1 complex (Ki = 80 nm) to atovaquone. When a L275F mutation was introduced into the yeast cytochrome b, the sensitivity of the yeast enzyme to atovaquone decreased (Ki = 100 nm) with no loss in activity, confirming that the L275F exchange contributes to the differential sensitivity of these two species to atovaquone. These results provide the first molecular description of how atovaquone binds to the bc1 complex and explain the differential inhibition of the fungal versus mammalian enzymes.

Members of the JNK pathway are organized together by virtue of interactions with JNK interacting protein 1 (JIP1), a scaffold protein. Here we have investigated the possibility that JIP1 may also affect the catalytic activity of Akt1, a serine/threonine kinase that has been implicated in multiple cellular processes, including survival and proliferation. JIP1 expression enhanced Akt1 kinase activity in a dose-dependent manner following serum starvation in 293 cells. Cellular activation of Akt1 following stimulation with low concentrations of insulin-like growth factor (IGF-1) was elevated in the presence of JIP1. JIP1 expression also prolonged Akt1 stimulation after a short IGF-1 pulse. The mechanism of JIP1-mediated Akt1 activation involved JIP1 protein binding to the Akt1 pleckstrin homology domain, which in turn promoted the phosphorylation of the activation T-loop of Akt1 by phosphoinositide-dependent kinase-1. These results suggest that, in certain cellular contexts, JIP1 may act as an Akt1 scaffold, which regulates the enzymatic activity of Akt1. This study also indicates that JIP1 expression can exert signaling effects independent of JNK activity

Gap junctions are intercellular channels formed by oligomerization of a protein called connexin (Cx). The heart expresses at least three connexin isotypes: Cx40, Cx43, and Cx45. A possible role for Cx40 in cardiac morphogenesis remains to be determined. We have characterized the anatomy and histology of fetal and newborn hearts obtained from crossing Cx40-deficient mice of mixed genetic background (C57BL/6x129Sv). Hearts were serial-sectioned (5 microm) along the coronal plane, stained with hematoxylin-eosin, and visualized by conventional light microscopy. Cardiac malformations in mice lacking Cx40 in one allele (Cx40+/-) included bifid atrial appendage, ventricular septal defect, tetralogy of Fallot (TOF), and an aortic arch abnormality. In Cx40-/- mice resulting from crossing of Cx40+/- mice, the most common cardiac malformations were double-outlet right ventricle (DORV), TOF, and endocardial cushion defects. Overall incidence of cardiac malformations was 6/33 (18%) in Cx40+/- mice and 4/12 (33%) in Cx40-/- mice. No cardiac malformations were observed in 15 wild-type mice studied. In addition, we examined 39 hearts from offspring of Cx40-/- matings. Frequency of cardiac malformations was even higher in this group (44%). Over one third of the hearts (14 of 39) showed conotruncal malformations corresponding to either DORV or TOF. Endocardial cushion defects were found in 3 out of 39 hearts. Our results suggest that Cx40 participates in cardiac morphogenesis, likely in association with other (unknown) products whose expression may vary with the genetic background of the mice

Bacterial GreA and GreB promote transcription elongation by stimulating an endogenous, endonucleolytic transcript cleavage activity of the RNA polymerase. The structure of Escherichia coli core RNA polymerase bound to GreB was determined by cryo-electron microscopy and image processing of helical crystals to a nominal resolution of 15 A, allowing fitting of high-resolution RNA polymerase and GreB structures. In the resulting model, the GreB N-terminal coiled-coil domain extends 45 A through a channel directly to the RNA polymerase active site. The model leads to detailed insights into the mechanism of Gre factor activity that explains a wide range of experimental observations and points to a key role for conserved acidic residues at the tip of the Gre factor coiled coil in modifying the RNA polymerase active site to catalyze the cleavage reaction. Mutational studies confirm that these positions are critical for Gre factor function.

Ras proteins regulate cellular growth and differentiation, and are mutated in 30% of cancers. We have shown recently that Ras is activated on and transmits signals from the Golgi apparatus as well as the plasma membrane but the mechanism of compartmentalized signalling was not determined. Here we show that, in response to Src-dependent activation of phospholipase Cgamma1, the Ras guanine nucleotide exchange factor RasGRP1 translocated to the Golgi where it activated Ras. Whereas Ca(2+) positively regulated Ras on the Golgi apparatus through RasGRP1, the same second messenger negatively regulated Ras on the plasma membrane by means of the Ras GTPase-activating protein CAPRI. Ras activation after T-cell receptor stimulation in Jurkat cells, rich in RasGRP1, was limited to the Golgi apparatus through the action of CAPRI, demonstrating unambiguously a physiological role for Ras on Golgi. Activation of Ras on Golgi also induced differentiation of PC12 cells, transformed fibroblasts and mediated radioresistance. Thus, activation of Ras on Golgi has important biological consequences and proceeds through a pathway distinct from the one that activates Ras on the plasma membrane

The node of Ranvier is a distinct domain of myelinated axons that is highly enriched in sodium channels and is critical for impulse propagation. During development, the channel subtypes expressed at the node undergo a transition from Nav1.2 to Nav1.6. Specialized junctions that form between the paranodal glial membranes and axon flank the nodes and are candidates to regulate their maturation and delineate their boundaries. To investigate these roles, we characterized node development in mice deficient in contactin-associated protein (Caspr), an integral junctional component. Paranodes in these mice lack transverse bands, a hallmark of the mature junction, and exhibit progressive disruption of axon-paranodal loop interactions in the CNS. Caspr mutant mice display significant abnormalities at central nodes; components of the nodes progressively disperse along axons, and many nodes fail to mature properly, persistently expressing Nav1.2 rather than Nav1.6. In contrast, PNS nodes are only modestly longer and, although maturation is delayed, eventually all express Nav1.6. Potassium channels are aberrantly clustered in the paranodes; these clusters are lost over time in the CNS, whereas they persist in the PNS. These findings indicate that interactions of the paranodal loops with the axon promote the transition in sodium channel subtypes at CNS nodes and provide a lateral diffusion barrier that, even in the absence of transverse bands, maintains a high concentration of components at the node and the integrity of voltage-gated channel domains

Combining structural data for the ribosome from x-ray crystallography and cryo-electron microscopy with dynamic models based on elastic network normal mode analysis, an atomically detailed picture of functionally important structural rearrangements that occur during translocation is elucidated. The dynamic model provides a near-atomic description of the ratchet-like rearrangement of the 70S ribosome seen in cryo-electron microscopy, and permits the identification of bridging interactions that either facilitate the conformational switching or maintain structural integrity of the 50S/30S interface. Motions of the tRNAs residing in the A and P sites also suggest the early stages of tRNA translocation as a result of this ratchet-like movement. Displacement of the L1 stalk, alternately closing and opening the intersubunit space near the E site, is observed in the dynamic model, in line with growing experimental evidence for the role of this structural component in facilitating the exiting of tRNA. Finally, a hinge-like transition in the 30S ribosomal subunit, similar to that observed in crystal structures of this complex, is also manifest as a dynamic mode of the ribosome. The coincidence of these dynamic transitions with the individual normal modes of the ribosome and the good correspondence between these motions and those observed in experiment suggest an underlying principle of nature to exploit the shape of molecular assemblies such as the ribosome to provide robustness to functionally important motions

The major facilitator superfamily represents the largest group of secondary membrane transporters in the cell. Here we report the 3.3 angstrom resolution structure of a member of this superfamily, GlpT, which transports glycerol-3-phosphate into the cytoplasm and inorganic phosphate into the periplasm. The amino- and carboxyl-terminal halves of the protein exhibit a pseudo two-fold symmetry. Closed off to the periplasm, a centrally located substrate-translocation pore contains two arginines at its closed end, which comprise the substrate-binding site. Upon substrate binding, the protein adopts a more compact conformation. We propose that GlpT operates by a single-binding site, alternating-access mechanism through a rocker-switch type of movement