Browsing by Author "Alqassim, Saif S"

Now showing 1 - 4 of 4

Functional Mimicry of Eukaryotic Actin Assembly by Pathogen Effector Proteins
(2022) Alqassim, Saif S
Abstract: The actin cytoskeleton lies at the heart of many essential cellular processes. There are hundreds of proteins that cells use to control the size and shape of actin cytoskeletal networks. As such, various pathogens utilize different strategies to hijack the infected eukaryotic host actin dynamics for their benefit. These include the control of upstream signaling pathways that lead to actin assembly, control of eukaryotic actin assembly factors, encoding toxins that distort regular actin dynamics, or by encoding effectors that directly interact with and assemble actin filaments. The latter class of effectors is unique in that, quite often, they assemble actin in a straightforward manner using novel sequences, folds, and molecular mechanisms. The study of these mechanisms promises to provide major insights into the fundamental determinants of actin assembly, as well as a deeper understanding of host–pathogen interactions in general, and contribute to therapeutic development efforts targeting their respective pathogens. This review discusses mechanisms and highlights shared and unique features of actin assembly by pathogen effectors that directly bind and assemble actin, focusing on eukaryotic actin nucleator functional mimics Rickettsia Sca2 (formin mimic), Burkholderia BimA (Ena/VASP mimic), and Vibrio VopL (tandem WH2-motif mimic).
Rickettsia Sca2 Recruits Two Actin Subunits for Nucleation but Lacks WH2 Domains.
(2019-02-05) Alqassim, Saif S
Abstract: The Rickettsia 1800-amino-acid auto transporter protein surface cell antigen 2 (Sca2) promotes actin polymerization on the surface of the bacterium to drive its movement using an actin comet-tail mechanism. Sca2 mimics eukaryotic for-mins in that it promotes both actin filament nucleation and elongation and competes with capping protein to generate filaments that are long and unbranched. However, despite these functional similarities, Sca2 is structurally unrelated to eukaryotic formins and achieves these functions through an entirely different mechanism. Thus, while formins are dimeric, Sca2 functions as amonomer. However, Sca2 displays intramolecular interactions and functional cooperativity between its N- and C-terminal do-mains that are crucial for actin nucleation and elongation. Here, we map the interaction of N- and C- terminal fragments ofSca2 and their contribution to actin binding and nucleation. We find that both the N- and C-terminal regions of Sca2 interact with actin monomers but only weakly, whereas the full-length protein binds two actin monomers with high affinity. Moreover, deletions at both ends of the N- and C-terminal regions disrupt their ability to interact with each other, suggesting that they form a contiguous ring-like structure that wraps around two actin subunits, analogous to the formin homology-2 domain. The discovery of Sca2 as an actin nucleator followed the identification of what appeared to be a repeat of three Wiskott-Aldrich syndrome homology 2 (WH2) domains in the middle of the molecule, consistent with the presence of WH2 domains in most actin nucleators. However, we show here that contrary to previous assumptions, Sca2 does not contain WH2 domains. Instead, our analysis indicates that the region containing the putative WH2 domains is folded as a globular domain that cooperates with other parts of the Sca2 molecule for actin binding and nucleation.
Single particle cryo-EM analysis of Rickettsia conorii Sca2 reveals a formin-like core
(2023) Alqassim, Saif S
Abstract: Spotted fever group Rickettsia undergo actin-based motility inside infected eukaryotic cells using Sca2 (surface cell antigen 2): an ~ 1800 amino-acid monomeric autotransporter protein that is surface-attached to the bacterium and responsible for the assembly of long unbranched actin tails. Sca2 is the only known functional mimic of eukaryotic formins, yet it shares no sequence similarities to the latter. Using structural and biochemical approaches we have previously shown that Sca2 uses a novel actin assembly mechanism. The first ~ 400 amino acids fold into helix-loop-helix repeats that form a crescent shape reminiscent of a formin FH2 monomer. Additionally, the N- and C- terminal halves of Sca2 display intramolecular interaction in an end-to-end manner and cooperate for actin assembly, mimicking a formin FH2 dimer. Towards a better structural understanding of this mechanism, we performed single-particle cryo-electron microscopy analysis of Sca2. While high-resolution structural details remain elusive, our model confirms the presence of a formin-like core: Sca2 indeed forms a doughnut shape, similar in diameter to a formin FH2 dimer and can accommodate two actin subunits. Extra electron density, thought to be contributed by the C-terminal repeat domain (CRD), covering one side is also observed. This structural analysis allows us to propose an updated model where nucleation proceeds by encircling two actin subunits, and elongation proceeds either by a formin-like mechanism that necessitates conformational changes in the observed Sca2 model, or via an insertional mechanism akin to that observed in the ParMRC system.
A tunable LIC1-adaptor interaction modulates dynein activity in a cargo-specific manner
(2020) Alqassim, Saif S
Abstract: Cytoplasmic dynein-1 (dynein) is the motor responsible for most retrograde transport of cargoes along microtubules in eukaryotic cells, including organelles, mRNA and viruses. Cargo selectivity and activation of processive motility depend on a group of so-called “activating adaptors” that link dynein to its general cofactor, dynactin, and cargoes. The mechanism by which these adaptors regulate dynein transport is poorly understood. Here, based on crystal structures, quantitative binding studies, and in vitro motility assays, we show that BICD2, CRACR2a, and HOOK3, representing three subfamilies of unrelated adaptors, interact with the same amphipathic helix of the dynein light intermediate chain-1 (LIC1). While the hydrophobic character of the interaction is conserved, the three adaptor subfamilies use different folds (coiled-coil, EF-hand, HOOK domain) and different surface contacts to bind the LIC1 helix with affinities ranging from 1.5 to 15.0 μM. We propose that a tunable LIC1-adaptor interaction modulates dynein’s motility in a cargo-specific manner.