X-ray and cryo-EM structures of monomeric and filamentous actin-like protein MamK reveal changes associated with polymerization

Abstract

Magnetotactic bacteria produce iron-rich magnetic nanoparticles that are enclosed by membrane invaginations to form magnetosomes so they are able to sense and act upon Earth's magnetic field. In Magnetospirillum and other magnetotactic bacteria, to combine their magnetic moments, magnetosomes align along filaments formed by a bacterial actin homolog, MamK. Here, we present the crystal structure of a nonpolymerizing mutant of MamK from Magnetospirillum magneticum AMB-1 at 1.8-Å resolution, revealing its close similarity to actin and MreB. The crystals contain AMPPNPbound monomeric MamK in two different conformations. To investigate conformational changes associated with polymerization, we used unmodified MamK protein and cryo-EM with helical 3D reconstruction in RELION to obtain a density map and a fully refined atomic model of MamK in filamentous form at 3.6-Å resolution. The filament is parallel (polar) double-helical, with a rise of 52.2 Å and a twist of 23.8°. As shown previously and unusually for actin-like filaments, the MamK subunits from each of the two strands are juxtaposed, creating an additional twofold axis along the filament. Compared with monomeric MamK, ADP-bound MamK in the filament undergoes a conformational change, rotating domains I and II against each other to further close the interdomain cleft between subdomains IB and IIB. The domain movement causes several loops to close around the nucleotide-binding pocket. Glu-143, a key residue for catalysis coordinating the magnesium ion, moves closer, presumably switching nucleotide hydrolysis upon polymerization-one of the hallmarks of cytomotive filaments of the actin type.magnetotactic bacteria | magnetosomes | filamentous proteins | bacterial cytoskeleton | cryo-EM I t was discovered in 1975 by Richard Blakemore that certain bacteria sense magnetic fields and swim according to the field's direction (1). The cells contain specialized iron-rich, membranebounded organelles, magnetosomes (2, 3). Magnetosomes contain magnetic nanoparticles that either together confer a magnetic moment to the bacterium or produce a signal that is used by the bacterium's chemotaxis machinery to swim in a particular direction.Magnetosomes occur in many different bacteria, but have been studied in some detail in gram-negative Magnetospirillum gryphiswaldense and Magnetospirillum magneticum AMB-1. In these organisms, typically 10 to 20 magnetosomes form as invaginations of the inner membrane, meaning that the nanoparticles are outside of the inner membrane, in the periplasm (4). They align along a straight line, into a chain, along the inner perimeter of the helical cells. Biomineralization and membrane invagination are somewhat coupled, although first empty or almost empty membrane invaginations form (5).In Magnetospirillum, magnetosome formation is encoded in gene clusters, included in a large magnetosome island. Many genes are required because the process of magnetosome formation is complex, requiring iron uptake, iron transport, biomineralization, membrane enclosure, m...

Cryo-EM structure of the bacterial actin AlfA reveals unique assembly and ATP-binding interactions and the absence of a conserved subdomain

“…The two strands are parallel and offset by a half-subunit stagger. The repeat distance is considerably shorter than found in other actins, which range from 512 to 834 Å (27)(28)(29)(30)(31)(32), yielding a highly twisted AlfA filament with only 8 subunits per turn of the two-start helix. The filament has a left-handed two-start twist, confirming our previous determination of handedness by tomographic reconstruction of negatively stained AlfA filaments (9).…”

Cryo-EM reconstruction of AlfA fromBacillus subtilisreveals the structure of a simplified actin-like filament at 3.4 Å resolution

“…The analysis was carried out with conventional, room temperature EM using negatively stained samples, which limited the resolution to around 15 Å. To obtain a detailed, near-atomic model of the filament structure, we turned to cryo-EM, which has recently shown much success in providing such information for actin, ParM, MamK and crenactin filaments (4,5,10,19).…”

“…The AlfA monomer structure shows a subdomain deletion and an unusual nucleotide-binding mode. For all previous actin-like filament structures determined by cryo-EM, reliable structures of the monomer obtained by X-ray crystallography were available to guide model building (4,5,10). With AlfA, such aid was not at hand, and our efforts to crystallise AlfA yielded only poorly diffracting crystals.…”

“…The roles that actins fulfil in cells reflect their ability to polymerise and are especially diverse in prokaryotes, where they function in cytokinesis, morphogenesis and DNA replication. Amongst the chromosomally encoded bacterial and archaeal actin-like proteins, we find: FtsA, an element of the cytokinetic (Z-) ring (3), MreB, a membrane-binding protein involved in the organisation of the cell wall synthesis and thus cell shape maintenance (2), as well as MamK, which organises the distribution of magnetosomes, iron-containing organelles found in magnetotactic bacteria (4). Within the archaeal phylum Crenarcheota, actin-like protein crenactin has been identified, and has been shown to be the closest so far in filament architecture and monomer fold to eukaryotic actin.…”

Crenactin forms actin-like double helical filaments regulated by arcadin-2

“…However, largely due to different contacts between strands, actin-like proteins evolved a wide variety of filament architectures. While actin forms right-handed, parallel and staggered filaments in all Eukaryotes (F-actin) (Holmes et al, 1990; von der Ecken et al, 2015), actin-like proteins differ in their filament architectures: MamK, required for magnetosome alignment in magnetotactic bacteria, forms right-handed filaments that have juxtaposed, non-staggered subunits (Bergeron et al, 2016; Löwe et al, 2016; Ozyamak et al, 2013a). ParM, making mitosis-like bipolar spindles during E. coli R1 plasmid segregation, produces left-handed, staggered filaments (Bharat et al, 2015; Gayathri et al, 2012).…”