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Albany 2019: 20th Conversation - Abstracts

category image Albany 2019
Conversation 20
June 11-15 2019
Adenine Press (2019)

Distance sensitive D-loop dynamics and near-atomic resolution of F-actin phalloidin structure by cryoEM

Actin is indispensable for eukaryotic cells. Therefore, molecular details of F-actin structure and dynamics are essential for our understanding of its key cellular functions. It is well established that nucleotide-bound state of F-actin (ATP/ADP-Pi or ADP) defines its dynamic properties, stability and interactions with regulatory factors. Previous studies also indicate that an innately flexible DNase I binding loop (D-loop, residues 40-50) plays a major role in such conformational dynamics. Recent advances in cryoEM provide high-resolution information about nucleotide bound states of actin, however the role of D loop in monomer to polymer transition still remains elusive. Intriguingly, phalloidin, a “gold standard” for actin staining in vivo and in vitro, is also known to stabilize actin filaments and affect the D-loop. Specifically, it can also convert polymerization-defective D loop mutants into stable polymers, thereby bypassing D loop dependency. By utilizing a multidisciplinary approach of mutational disulphide crosslinking, light scattering measurements and cryoEM we probe the structural mechanisms that govern D-loop transitions in actin dynamics and how phalloidin stabilizes F-actin. Our biochemical data provides a molecular ruler based model of how intraprotomer distance between two D-loop residues facilitates a transition from G to F-actin and vice versa. Additionally, we report the first 3.7 Å resolution structure of Phalloidin bound F-actin in the ADP-Pi state. Our structure supports (i) the role of methylation of His 73 on actin in Pi binding, (ii) shows that phalloidin inhibits Pi release from actin filaments via their direct interaction, (iii) defines phalloidin binding site on F-actin filaments and reveals how it restricts the relative movement between the two protofilaments. Together, our results provide new molecular details of F-actin structure and D loop dynamics. This structural and functional information may be also useful for designing phalloidin derivatives and selecting best phalloidin matching conjugate for staining different actin forms and structures. sanchita-fig-1ab.png

Fig. 1: CryoEM structure of ADP-BeFx-F-actin-phalloidin
(A) Overall structure of ADP-BeFx-F-actin-phalloidin at 3.7 Å resolution, fitted with its atomic model. Red characters mark the relative position of actin subunits in the filament.(B)Representative regions (marked by amino acid numbers) of the density map fitted with their atomic models, showing the quality of the map.

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Fig2: Phalloidin can rescue the polymerization of oxidized double D-loop mutant F-C41C45.
(A) Yeast actin mutant C41C45, with two cyteines in the D-loop, was polymerized in the presence of 2mM MgCl2 as monitored by light scattering. Upon polymerization 20 uM CuSO4 was added to catalyze the disulfide bond formation between C41 and C45. Finally, the polymerization of destructed filaments were rescued by the addition of equimolar phalloidin. Arrows indicate the described additions to the actin sample. (B)Electron micrographs of F-C41C45 before and after oxidation. Electron microscopy samples were taken from experiments ran in parallel to the one shown in A. For control filaments, 10mM TCEP was added to ensure disulfide bond reduction. The scale bar in control image represents 0.2µm.

Sanchaita Das1*,
Peng Ge3, 4*,
Zeynep A. Oztug Durer1*,
Elena E. Grintsevich1,
Z. Hong Zhou3, 4, and
Emil Reisler1, 2

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Sanchaita obtained her PhD in 2011 from Wesleyan University under Dr. Donald Oliver. Then she did post-doctoral research with Dr. Joachim Frank, Columbia , Steven Doxsey and David Lambright, Univ of Mass. Currently she is an assistant project scientist with Dr. Emil Reisler at UCLA, and will present a short oral.Sanchaita can be contacted by email (sanchaita06@gmail.com) or phone (8606044804).

1Department of Chemistry and Biochemistry, UCLA, Los Angeles, California, USA
2Molecular Biology Institute, UCLA, Los Angeles, California, USA
3Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles (UCLA), Los Angeles, California, USA
4California NanoSystems Institute (CNSI), UCLA, Los Angeles, California, USA
*contributed equally
Corresponding author: Emil Reisler. reisler@mbi.ucla.edu