Mechanochemical Coupling in the Myosin Motor Domain. II. Analysis of Critical Residues

An important challenge in the analysis of mechanochemical coupling in molecular motors is to identify residues that dictate the tight coupling between the chemical site and distant structural rearrangements. In this work, a systematic attempt is made to tackle this issue for the conventional myosin. By judiciously combining a range of computational techniques with different approximations and strength, which include targeted molecular dynamics, normal mode analysis, and statistical coupling analysis, we are able to identify a set of important residues and propose their relevant function during the recovery stroke of myosin. These analyses also allowed us to make connections with previous experimental and computational studies in a critical manner. The behavior of the widely used reporter residue, Trp501, in the simulations confirms the concern that its fluorescence does not simply reflect the relay loop conformation or active-site open/close but depends subtly on its microenvironment. The findings in the targeted molecular dynamics and a previous minimum energy path analysis of the recovery stroke have been compared and analyzed, which emphasized the difference and complementarity of the two approaches. In conjunction with our previous studies, the current set of investigations suggest that the modulation of structural flexibility at both the local (e.g., active-site) and domain scales with strategically placed “hotspot” residues and phosphate chemistry is likely the general feature for mechanochemical coupling in many molecular motors. The fundamental strategies of examining both collective and local changes and combining physically motivated methods and informatics-driven techniques are expected to be valuable to the study of other molecular motors and allosteric systems in general

Integration and isolation in the global petrochemical industry: A multi-scalar corporate network analysis

The global petrochemical industry has long been characterized by stable patterns of Western corporate and geographic leadership, but since the early 2000s, the global playing field has changed significantly. China has overtaken the United States and Europe as the world’s largest petrochemical producer, and other emerging economies have emerged as global petrochemical players. Combining insights from scholarship on global corporate elites, world city networks, and relational economic geography, this article examines patterns in the corporate networks of leading petrochemical corporations. The research is based on a multi-scalar corporate network analysis, applying social network analysis to identify board interlocks, joint venture interlocks, and spatial interlocks between corporations. Through analyzing corporate networks across multiple scales, the research reveals patterns of both integration and isolation within the petrochemical industry. Isolation is evident in disconnected regional corporate elite networks, where the established North Atlantic corporate elite is interconnected through board interlocks, while corporate networks in Asia and other emerging economies remain disconnected. However, high levels of integration within the industry are also evident in an interconnected international company system formed through joint venture collaborations, and in overlapping subsidiary networks centered on petrochemical hubs around the world. The article argues that the results demonstrate a combination of resilience and change, or path dependence and contingency, in patterns of corporate power and collaboration. Western company networks still form the social and spatial backbone of the industry, but these have been challenged by emerging strategic centers and isolated elite networks in other parts of the world. This paper contributes to debates on industrial corporate elites, multiple globalizations, and the multipolar global economy

The C. elegans Opa1 Homologue EAT-3 Is Essential for Resistance to Free Radicals

The C. elegans eat-3 gene encodes a mitochondrial dynamin family member homologous to Opa1 in humans and Mgm1 in yeast. We find that mutations in the C. elegans eat-3 locus cause mitochondria to fragment in agreement with the mutant phenotypes observed in yeast and mammalian cells. Electron microscopy shows that the matrices of fragmented mitochondria in eat-3 mutants are divided by inner membrane septae, suggestive of a specific defect in fusion of the mitochondrial inner membrane. In addition, we find that C. elegans eat-3 mutant animals are smaller, grow slower, and have smaller broodsizes than C. elegans mutants with defects in other mitochondrial fission and fusion proteins. Although mammalian Opa1 is antiapoptotic, mutations in the canonical C. elegans cell death genes ced-3 and ced-4 do not suppress the slow growth and small broodsize phenotypes of eat-3 mutants. Instead, the phenotypes of eat-3 mutants are consistent with defects in oxidative phosphorylation. Moreover, eat-3 mutants are hypersensitive to paraquat, which promotes damage by free radicals, and they are sensitive to loss of the mitochondrial superoxide dismutase sod-2. We conclude that free radicals contribute to the pathology of C. elegans eat-3 mutants

Faculty Mentoring

3 online resources (PDF files): Final report, Poster presentation, and Project summaryAs the University of Minnesota seeks to become one of the top three public universities in the world, we need to be attentive to issues concerning the life-course of faculty to support faculty success at all points of their careers. The Strategic Positioning Task Force on Faculty Culture, in their final report released in May, pointed to faculty mentoring as a key strategy to move us in the direction of excellence. In addition, the Provost's recent memo to department chairs regarding the revision of departmental standards for tenure and promotion (7.12 statements) requires a statement about how senior faculty will mentor junior assistant and associate faculty.President's Emerging Leadership ProgramDouah, Remi; Letawsky Shultz, Nikki; Nackerud, Shane; Radcliffe, Peter M.; Reubold, Todd. (2007). Faculty Mentoring. Retrieved from the University Digital Conservancy, https://hdl.handle.net/11299/5487

Crystal structure of the tegument protein UL82 (pp71) from human cytomegalovirus

Human cytomegalovirus (HCMV) is an opportunistic pathogen that infects a majority of the world population. It may cause severe disease in immunocompromised people and lead to pregnancy loss or grave disabilities of the fetus upon congenital infection. For effective replication and lifelong persistence in its host, HCMV relies on diverse functions of its tegument protein UL82, also known as pp71. Up to now, little is known about the molecular mechanisms underlying the multiple functions of this crucial viral protein. Here, we describe the X-ray structure of full-length UL82 to a resolution of 2.7 Å. A single polypeptide chain of 559 amino acids mainly folds into three ß-barrels. We show that UL82 forms a dimer in the crystal as well as in solution. We identify point mutations that disturb the dimerization interface and show that the mutant protein is monomeric in solution and upon expression in human cells. On the basis of the three-dimensional structure, we identify structural homologs of UL82 from other herpesviruses and analyze whether their functions are preserved in UL82. We demonstrate that UL82, despite its structural homology to viral deoxyuridinetriphosphatases (dUTPases), does not possess dUTPase activity. Prompted by the structural homology of UL82 to the ORF10 protein of murine herpesvirus 68 (MHV68), which is known to interact with the RNA export factor ribonucleic acid export 1 (Rae1), we performed coimmunoprecipitations and demonstrated that UL82 indeed interacts with Rae1. This suggests that HCMV UL82 may play a role in mRNA export from the nucleus similar to ORF10 encoded by the gammaherpesviruses MHV68

Toward understanding actin activation of myosin ATPase: The role of myosin surface loops

To understand the complicated interplay when a traveling myosin head reaches interaction distance with two actins in a filament we looked to three myosin loops that early on exert their influences from the “outside” of the myosin. On these we conduct, functionally test, and interpret strategically chosen mutations at sites thought from crystallography to be a patch for binding the “first” of the two actins. One loop bears a hydrophobic triplet of residues, one is the so-called “loop 2,” and the third is the “cardiomyopathy” loop. So far as we know, the myosin sites that first respond are the two lysine-rich loops that produce an ionic strength-dependent weak-binding complex with actin. Subsequently, the three loops of interest bind the first actin simultaneously, and all three assist in closing the cleft in the 50-kDa domain of the myosin, a closure that results in transition from weak to strong binding and precedes rapid P(i) release and motility. Mutational analysis shows that each such loop contact is distinctive in the route by which it communicates with its specific target elsewhere in myosin. The strongest contact with actin, for example, is that of the triplet-bearing loop. On the other hand, that of loop 2 (dependent on drawing close two myosin lysines and two actin aspartates) is probably responsible for opening switch I and uncovering the γ-phosphate moiety of bound ATP. Taking into account these findings, we begin to arrange in order many molecular events in muscle function

Crystal structure of the GTPase domain of rat dynamin 1

Here, we present the 1.9-Å crystal structure of the nucleotide-free GTPase domain of dynamin 1 from Rattus norvegicus. The structure corresponds to an extended form of the canonical GTPase fold observed in Ras proteins. Both nucleotide-binding switch motifs are well resolved, adopting conformations that closely resemble a GTP-bound state not previously observed for nucleotide-free GTPases. Two highly conserved arginines, Arg-66 and Arg-67, greatly restrict the mobility of switch I and are ideally positioned to relay information about the nucleotide state to other parts of the protein. Our results support a model in which switch I residue Arg-59 gates GTP binding in an assembly-dependent manner and the GTPase effector domain functions as an assembly-dependent GTPase activating protein in the fashion of RGS-type GAPs

Repriming the actomyosin crossbridge cycle

The central features of the mechanical cycle that drives the contraction of muscle are two translational steps: the working stroke, whereby an attached myosin crossbridge moves relative to the actin filament, and the repriming step, in which the crossbridge returns to its original orientation. Although the mechanism of the first of these is understood in some detail, that of the second has received less attention. Here, we show that repriming occurs after detachment of the crossbridge from the actin, rather than intervening between two actomyosin states with ATP bound [Eisenberg, E. & Greene, L. E. (1980) Annu. Rev. Physiol. 42, 293–309]. To discriminate between these two models we investigated the single-molecule mechanics of the myosin–actin interaction in the presence of ATP analogues such as GTP, for which the hydrolytic step itself limits the actomyosin GTPase rate to a much lower rate than for ATP. The lifetimes of bound states was proportional to 1/[GTP], indicating that during the bound period myosin was in the actomyosin rigor configuration. Moreover, despite the very low actomyosin GTPase, the rate of actin binding and formation of the rigor state was higher than with ATP; it follows that most interactions with actin result in the release of GTP and not of the products, GDP and phosphate. There was no significant movement of the actin during this interaction, so repriming must occur while myosin is dissociated, as in the original Lymn–Taylor scheme [Lymn, R. W. & Taylor, E. W. (1971) Biochemistry 10, 4617–4624]