Advances in structure determination by cryo‐EM to unravel membrane‐spanning pore formation

Abstract

The beta pore‐forming proteins (β‐PFPs) are a large class of polypeptides that are produced by all Kingdoms of life to contribute to their species' own survival. Pore assembly is a sophisticated multi‐step process that includes receptor/membrane recognition and oligomerization events, and is ensued by large‐scale structural rearrangements, which facilitate maturation of a prepore into a functional membrane spanning pore. A full understanding of pore formation, assembly, and maturation has traditionally been hindered by a lack of structural data; particularly for assemblies representing differing conformations of functional pores. However, recent advancements in cryo‐electron microscopy (cryo‐EM) techniques have provided the opportunity to delineate the structures of such flexible complexes, and in different states, to near‐atomic resolution. In this review, we place a particular emphasis on the use of cryo‐EM to uncover the mechanistic details including architecture, activation, and maturation for some of the prominent members of this family.

Structural and functional insights into the first Bacillus thuringiensis vegetative insecticidal protein of the Vpb4 fold, active against western corn rootworm

“…Analysis of the Vpb4Da2 structure determined by X-ray crystallography revealed a protein rich in β-strands exhibiting a six-domain architecture. Vpb4Da2 has an overall structural homology to the Bacterial_exotoxin_B family of the β-barrel pore-forming proteins (β-PFPs) [ 21 , 47 , 48 ], namely the structural region spanning domains 1–3 of the B . anthracis PA protein [ 16 ] and that of the C .…”

“…Proteins from the Bacterial_exotoxin_B family are non-toxic binding components (B-component) of the AB-toxin super-family [ 15 ] and include protective antigen (PA) [ 16 ], C2 component (C2-II) [ 17 ], Iota toxin component (Ib-component) [ 18 ], vegetative insecticidal protein 1, (Vpb1; formerly known as Vip1) [ 19 ] and other Vpb4’s [ 20 ]. The amino-terminal domains 1–3 of these proteins exhibit a conserved structural organization shared across the family whereas the carboxyl-terminal is comprised of at least one domain [ 21 ]. For PA, domain 1 contains a cysteine protease cleavage site integral to oligomerization and pre-pore formation, domain 2 contains a long stem region that changes conformation at acidic pH to form a β-barrel pore, and domain 3 is involved in oligomer formation [ 22 ].…”

Using NMR Chemical Shifts and Cryo-EM Density Restraints in Iterative Rosetta-MD Protein Structure Refinement

“…In NMR spectroscopy, distance restraints of proximal atoms are obtained and have been utilized to elucidate almost 13 000 protein structures . Cryo-electron microscopy (cryo-EM) is another increasingly popular technique to determine macromolecular structures. − Cryo-EM is continuing to revolutionize the field of structural biology and was also worthy of a recent noble prize award. ,, …”