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We are making the the manuscripts listed on this page available to the academic community for use in teaching and research. The copyrights associated with each paper remain with the appropriate parties.

201920182017 – 2016

2019

Kaur H, Jamalidinan F, Condon SGF, Senes A, Hoskins AA

Analysis of spliceosome dynamics by maximum likelihood fitting of dwell time distributions

Methods 2019 153, 13-21

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Colocalization single-molecule methods can provide a wealth of information concerning the ordering and dynamics of biomolecule assembly. These have been used extensively to study the pathways of spliceosome assembly in vitro. Key to these experiments is the measurement of binding times-either the dwell times of a multi-molecular interaction or times in between binding events. By analyzing hundreds of these times, many new insights into the kinetic pathways governing spliceosome assembly have been obtained. Collections of binding times are often plotted as histograms and can be fit to kinetic models using a variety of methods. Here, we describe the use of maximum likelihood methods to fit dwell time distributions without binning. In addition, we discuss several aspects of analyzing these distributions with histograms and pitfalls that can be encountered if improperly binned histograms are used. We have automated several aspects of maximum likelihood fitting of dwell time distributions in the AGATHA software package.

2018

Niemann MCE, Weber H, Hluska T, Leonte G, Anderson SM, Novak O, Senes A, Werner T

The cytokinin oxidase/dehydrogenase CKX1 is a membrane-bound protein requiring homooligomerization in the endoplasmic reticulum for its cellular activity

Plant Physiology 2018 176, 2024-39

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Degradation of the plant hormone cytokinin is controlled by cytokinin oxidase/dehydrogenase (CKX) enzymes. The molecular and cellular behavior of these proteins is still largely unknown. In this study, we show that CKX1 is a type-II single-pass membrane protein that predominantly localizes to the endoplasmic reticulum (ER). This indicates that this CKX isoform is a bona fide ER protein directly controlling the cytokinin which triggers the signaling from the ER. By using various approaches, we demonstrate that CKX1 forms homodimers and homooligomers in vivo. The N-terminal part of CKX1 was necessary and sufficient for the protein oligomerization as well as for targeting and retention in the ER. Moreover, we show that protein-protein interaction is largely facilitated by transmembrane helices and depends on a functional GxxxG-like interaction motif. Importantly, mutations rendering CKX1 monomeric interfere with its steady-state localization in the ER and cause a loss of the CKX1 biological activity by increasing its ER-associated degradation. Therefore, our study provides evidence that oligomerization is a crucial parameter regulating CKX1 biological activity and the cytokinin concentration in the ER. The work also lends a strong support for the cytokinin signaling from the ER and for the functional relevance of the cytokinin pool in this compartment.

Condon SGF*, Mahbuba DA*, Armstrong CR, Diaz-Vazquez G, Craven SJ, LaPointe LM, Khadria AS, Chadda R, Crooks JA, Rangarajan N, Weibel DB, Hoskins AA, Robertson JL, Cui Q, Senes A

The FtsLB sub-complex of the bacterial divisome is a tetramer with an uninterrupted FtsL helix linking the transmembrane and periplasmic regions

J. Biol. Chem. 2018 293, 1623-41 (*authors contributed equally)

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In Escherichia coli, FtsLB plays a central role in the initiation of cell division, possibly transducing a signal that will eventually lead to the activation of peptidoglycan remodeling at the forming septum. The molecular mechanisms by which FtsLB operates in the divisome, however, are not understood. Here we present a structural analysis of the FtsLB complex — performed with biophysical, computational and in vivo methods — that establishes the organization of the transmembrane region and proximal coiled coil of the complex. FRET analysis in vitro is consistent with formation of a tetramer composed of two FtsL and two FtsB subunits. We predicted subunit contacts through co-evolutionary analysis and used them to compute a structural model of the complex. The transmembrane region of FtsLB is stabilized by hydrophobic packing and by a complex network of hydrogen bonds. The coiled coil domain likely terminates near the critical Constriction Control Domain, which might correspond to a structural transition. Presence of strongly polar amino acids within the core of the tetrameric coiled coil suggests that the coil may split into two independent FtsQ-binding domains. The helix of FtsB is interrupted between the transmembrane and coiled coil regions by a flexible Gly-rich linker. Conversely, the data suggest that FtsL forms an uninterrupted helix across the two regions, and that integrity of this helix is indispensable for the function of the complex. The FtsL helix is thus a candidate for acting as a potential mechanical connection to communicate conformational changes between periplasmic, membrane, and cytoplasmic regions.

2017

Anderson SM*, Mueller BK*, Lange EJ and Senes A

Combination of Cα-H hydrogen bonds and van der Waals packing modulates the stability of GxxxG-mediated dimers in membranes

J Am Chem Soc. (2017) 139, 15774-83 (*authors contributed equally)

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Abstract: The GxxxG motif is frequently found at the dimerization interface of a transmembrane structural motif called GASright, which is characterized by a short interhelical distance and a right-handed crossing angle between the helices. In GASright dimers, such as glycophorin A (GpA), BNIP3, and members of the ErbB family, the backbones of the helices are in contact, and they invariably display networks of 4 to 8 weak hydrogen bonds between Cα-H carbon donors and carbonyl acceptors on opposing helices (Cα-H···O=C hydrogen bonds). These networks of weak hydrogen bonds at the helix-helix interface are presumably stabilizing, but their energetic contribution to dimerization has yet to be determined experimentally. Here, we present a computational and experimental structure-based analysis of GASright dimers of different predicted stabilities, which show that a combination of van der Waals packing and Cα-H hydrogen bonding predicts the experimental trend of dimerization propensities. This finding provides experimental support for the hypothesis that the networks of Cα-H hydrogen bonds are major contributors to the free energy of association of GxxxG-mediated dimers. The structural comparison between groups of GASright dimers of different stabilities reveals distinct sequence as well as conformational preferences. Stability correlates with shorter interhelical distances, narrower crossing angles, better packing, and the formation of larger networks of Cα-H hydrogen bonds. The identification of these structural rules provides insight on how nature could modulate stability in GASright and finely tune dimerization to support biological function.

Guo X, Niemi NM, Hutchins PD, Condon SGF, Jochem A, Ulbrich A, Higbee AJ, Russell JD, Senes A, Coon JJ, and Pagliarini DJ

“Ptc7p dephosphorylates select mitochondrial proteins to enhance metabolic function”

Cell Reports (2017) 18, 307-13

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Proper maintenance of mitochondrial activity is essential for metabolic homeostasis. Widespread phosphorylation of mitochondrial proteins may be an important element of this process; yet little is known about which enzymes control mitochondrial phosphorylation, or which phosphosites have functional impact. We investigated these issues by disrupting Ptc7p — a conserved but largely uncharacterized mitochondrial matrix PP2C-type phosphatase. Loss of Ptc7p caused respiratory growth defects concomitant with elevated phosphorylation of select matrix proteins. Among these, Δptc7 yeast exhibited an increase in phosphorylation of Cit1p — the canonical citrate synthase of the tricarboxylic acid cycle — that diminished its activity. We found that phosphorylation of S462 can eliminate Cit1p enzymatic activity likely by disrupting its proper dimerization, and that Ptc7p-driven dephosphorylation rescued Cit1p activity. Collectively, our work connects Ptc7p to an essential TCA cycle function and to additional phosphorylation events that may affect mitochondrial activity inadvertently or in a regulatory manner

2016

Cover of Armstrong Biochim. Biophys. Acta 2016 1858, 2573-83

Armstrong CR, Senes A

Screening for transmembrane association in divisome proteins using TOXGREEN, a high-throughput variant of the TOXCAT assay”

Biochim. Biophys. Acta 2016 1858, 2573-83

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TOXCAT is a widely used genetic assay to study interactions of transmembrane helices within the inner membrane of the bacterium Escherichia coli. TOXCAT is based on a fusion construct that links a transmembrane domain of interest with a cytoplasmic DNA-binding domain from the Vibrio cholerae ToxR protein. Interaction driven by the transmembrane domain results in dimerization of the ToxR domain, which, in turn, activates the expression of the reporter gene chloramphenicol acetyl transferase (CAT). Quantification of CAT is used as a measure of the ability of the transmembrane domain to self-associate. Because the quantification of CAT is relatively laborious, we developed a high-throughput variant of the assay, TOXGREEN, based on the expression of super-folded GFP and detection of fluorescence directly in unprocessed cell cultures. Careful side-by-side comparison of TOXCAT and TOXGREEN demonstrates that the methods have comparable response, dynamic range, sensitivity and intrinsic variability both in LB and minimal media. The greatly enhanced workflow makes TOXGREEN much more scalable and ideal for screening, since hundreds of constructs can be rapidly assessed in 96 well plates. Even for small scale investigations, TOXGREEN significantly reduces time, labor and cost associated with the procedure. We demonstrate applicability with a large screening for self-association among the transmembrane domains of bitopic proteins of the divisome (FtsL, FtsB, FtsQ, FtsI, FtsN, ZipA and EzrA) belonging to 11 bacterial species. The analysis confirms a previously reported tendency for FtsB to self-associate, and suggests that the transmembrane domains of ZipA, EzrA and FtsN may also possibly oligomerize.

Cover of Barth Nat Struct Mol Biol 2016

Barth P, Senes A

Toward high-resolution computational design of the structure and function of helical membrane proteins

Nat Struct Mol Biol 2016 23, 475-80

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The computational design of α-helical membrane proteins is still in its infancy but has already made great progress. De novo design allows stable, specific and active minimal oligomeric systems to be obtained. Computational reengineering can improve the stability and function of naturally occurring membrane proteins. Currently, the major hurdle for the field is the experimental characterization of the designs. The emergence of new structural methods for membrane proteins will accelerate progress.

Cover of Zhang EMBO J 2016

Zhang Z, Subramaniam S, Kale J, Liao C, Huang B, Brahmbhatt H, Condon SGF, Lapolla SM, Hays, FA, Ding J, He F, Zhang XC, Li J, Senes A, Andrews DW, Lin J

BH3-in-groove dimerization initiates and helix 9 dimerization expands Bax pore assembly in membranes

EMBO J 2016 35, 208-36

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Pro-apoptotic Bax induces mitochondrial outer membrane permeabilization (MOMP) by forming oligomers through a largely undefined process. Using site-specific disulfide crosslinking, compartment-specific chemical labeling, and mutational analysis, we found that activated integral membrane Bax proteins form a BH3-in-groove dimer interface on the MOM surface similar to that observed in crystals. However, after the α5 helix was released into the MOM, the remaining interface with α2, α3, and α4 helices was rearranged. Another dimer interface was formed inside the MOM by two intersected or parallel α9 helices. Combinations of these interfaces generated oligomers in the MOM. Oligomerization was initiated by BH3-in-groove dimerization, without which neither the other dimerizations nor MOMP occurred. In contrast, α9 dimerization occurred downstream and was required for release of large but not small proteins from mitochondria. Moreover, the release of large proteins was facilitated by α9 insertion into the MOM and localization to the pore rim. Therefore, the BH3-in-groove dimerization on the MOM nucleates the assembly of an oligomeric Bax pore that is enlarged by α9 dimerization at the rim.