Structural plasticity of bacterial ESCRT-III protein PspA in higher-order assemblies.

Eukaryotic members of the endosome sorting complex required for transport-III (ESCRT-III) family have been shown to form diverse higher-order assemblies. The bacterial phage shock protein A (PspA) has been identified as a member of the ESCRT-III superfamily, and PspA homo-oligomerizes to form rod-shaped assemblies. As observed for eukaryotic ESCRT-III, PspA forms tubular assemblies of varying diameters. Using electron cryo-electron microscopy, we determined 61 Synechocystis PspA structures and observed in molecular detail how the structural plasticity of PspA rods is mediated by conformational changes at three hinge regions in the monomer and by the fixed and changing molecular contacts between protomers. Moreover, we reduced and increased the structural plasticity of PspA rods by removing the loop connecting helices α3/α4 and the addition of nucleotides, respectively. Based on our analysis of PspA-mediated membrane remodeling, we suggest that the observed mode of structural plasticity is a prerequisite for the biological function of ESCRT-III members.

SynDLP is a dynamin-like protein of Synechocystis sp. PCC 6803 with eukaryotic features.

Dynamin-like proteins are membrane remodeling GTPases with well-understood functions in eukaryotic cells. However, bacterial dynamin-like proteins are still poorly investigated. SynDLP, the dynamin-like protein of the cyanobacterium Synechocystis sp. PCC 6803, forms ordered oligomers in solution. The 3.7 Å resolution cryo-EM structure of SynDLP oligomers reveals the presence of oligomeric stalk interfaces typical for eukaryotic dynamin-like proteins. The bundle signaling element domain shows distinct features, such as an intramolecular disulfide bridge that affects the GTPase activity, or an expanded intermolecular interface with the GTPase domain. In addition to typical GD-GD contacts, such atypical GTPase domain interfaces might be a GTPase activity regulating tool in oligomerized SynDLP. Furthermore, we show that SynDLP interacts with and intercalates into membranes containing negatively charged thylakoid membrane lipids independent of nucleotides. The structural characteristics of SynDLP oligomers suggest it to be the closest known bacterial ancestor of eukaryotic dynamin.

Membrane destabilization and pore formation induced by the Synechocystis IM30 protein.

The inner membrane-associated protein of 30 kDa (IM30) is essential in chloroplasts and cyanobacteria. The spatio-temporal cellular localization of the protein appears to be highly dynamic and triggered by internal as well as external stimuli, mainly light intensity. The soluble fraction of the protein is localized in the cyanobacterial cytoplasm or the chloroplast stroma, respectively. Additionally, the protein attaches to the thylakoid membrane as well as to the chloroplast inner envelope or the cyanobacterial cytoplasmic membrane, respectively, especially under conditions of membrane stress. IM30 is involved in thylakoid membrane biogenesis and/or maintenance, where it either stabilizes membranes and/or triggers membrane-fusion processes. These apparently contradicting functions have to be tightly controlled and separated spatiotemporally in chloroplasts and cyanobacteria. IM30's fusogenic activity depends on Mg2+ binding to IM30; yet, it still is unclear how Mg2+-loaded IM30 interacts with membranes and promotes membrane fusion. Here, we show that the interaction of Mg2+ with IM30 results in increased binding of IM30 to native, as well as model, membranes. Via atomic force microscopy in liquid, IM30-induced bilayer defects were observed in solid-supported bilayers in the presence of Mg2+. These structures differ dramatically from the membrane-stabilizing carpet structures that were previously observed in the absence of Mg2+. Thus, Mg2+-induced alterations of the IM30 structure switch the IM30 activity from a membrane-stabilizing to a membrane-destabilizing function, a crucial step in membrane fusion.

Human Claudin-7 cis-Interactions Are Not Crucial for Membrane-Membrane (Trans-) Interactions.

Human Claudin-7 (Cldn7) is a member of the Claudin (Cldn) superfamily. In vivo, these proteins form tight junctions, which establish constricted connections between cells. Cldns oligomerize within the membrane plane (= cis-interaction), and also interact with Cldns from adjacent cells (= trans-interaction). Interactions of Cldns are typically studied in vivo and structural analyses of isolated Cldns are limited. Here, we describe heterologous expression in E. coli and purification of human Cldn7, enabling in vitro analyses of the isolated protein using detergent and model membrane systems. Cldn7 exists as a monomer, hexamer, and various higher oligomers in micelles. While only limited unfolding of the protein was observed in the presence of the anionic detergent sodium dodecyl sulfate, decreased ionic strength did affect Cldn7 cis-interactions. Furthermore, we identified two amino acids which mediate electrostatic cis-interactions and analyzed the impact of disturbed cis-interaction on trans-contacts via atomic force microscopy and monitoring Förster resonance energy transfer between fluorescently labeled Cldn7-containing proteoliposomes. Our results indicate that Cldn7 cis-oligomerization might not be a prerequisite for establishing trans-contacts.

PspA adopts an ESCRT-III-like fold and remodels bacterial membranes.

PspA is the main effector of the phage shock protein (Psp) system and preserves the bacterial inner membrane integrity and function. Here, we present the 3.6 Å resolution cryoelectron microscopy (cryo-EM) structure of PspA assembled in helical rods. PspA monomers adopt a canonical ESCRT-III fold in an extended open conformation. PspA rods are capable of enclosing lipids and generating positive membrane curvature. Using cryo-EM, we visualized how PspA remodels membrane vesicles into µm-sized structures and how it mediates the formation of internalized vesicular structures. Hotspots of these activities are zones derived from PspA assemblies, serving as lipid transfer platforms and linking previously separated lipid structures. These membrane fusion and fission activities are in line with the described functional properties of bacterial PspA/IM30/LiaH proteins. Our structural and functional analyses reveal that bacterial PspA belongs to the evolutionary ancestry of ESCRT-III proteins involved in membrane remodeling.

Binding and/or hydrolysis of purine-based nucleotides is not required for IM30 ring formation.

IM30, the inner membrane-associated protein of 30 kDa, is conserved in cyanobacteria and chloroplasts. Although its exact physiological function is still mysterious, IM30 is clearly essential for thylakoid membrane biogenesis and/or dynamics. Recently, a cryptic IM30 GTPase activity has been reported, albeit thus far no physiological function has been attributed to this. Yet, it is still possible that GTP binding/hydrolysis affects formation of the prototypical large homo-oligomeric IM30 ring and rod structures. Here, we show that the Synechocystis sp. PCC 6803 IM30 protein in fact is an NTPase that hydrolyzes GTP and ATP, but not CTP or UTP, with about identical rates. While IM30 forms large oligomeric ring complexes, nucleotide binding and/or hydrolysis are clearly not required for ring formation.

IM30 IDPs form a membrane-protective carpet upon super-complex disassembly.

Members of the phage shock protein A (PspA) family, including the inner membrane-associated protein of 30 kDa (IM30), are suggested to stabilize stressed cellular membranes. Furthermore, IM30 is essential in thylakoid membrane-containing chloroplasts and cyanobacteria, where it is involved in membrane biogenesis and/or remodeling. While it is well known that PspA and IM30 bind to membranes, the mechanism of membrane stabilization is still enigmatic. Here we report that ring-shaped IM30 super-complexes disassemble on membranes, resulting in formation of a membrane-protecting protein carpet. Upon ring dissociation, the C-terminal domain of IM30 unfolds, and the protomers self-assemble on membranes. IM30 assemblies at membranes have been observed before in vivo and were associated with stress response in cyanobacteria and chloroplasts. These assemblies likely correspond to the here identified carpet structures. Our study defines the thus far enigmatic structural basis for the physiological function of IM30 and related proteins, including PspA, and highlights a hitherto unrecognized concept of membrane stabilization by intrinsically disordered proteins.

Proton Leakage Is Sensed by IM30 and Activates IM30-Triggered Membrane Fusion.

The inner membrane-associated protein of 30 kDa (IM30) is crucial for the development and maintenance of the thylakoid membrane system in chloroplasts and cyanobacteria. While its exact physiological function still is under debate, it has recently been suggested that IM30 has (at least) a dual function, and the protein is involved in stabilization of the thylakoid membrane as well as in Mg2+-dependent membrane fusion. IM30 binds to negatively charged membrane lipids, preferentially at stressed membrane regions where protons potentially leak out from the thylakoid lumen into the chloroplast stroma or the cyanobacterial cytoplasm, respectively. Here we show in vitro that IM30 membrane binding, as well as membrane fusion, is strongly increased in acidic environments. This enhanced activity involves a rearrangement of the protein structure. We suggest that this acid-induced transition is part of a mechanism that allows IM30 to (i) sense sites of proton leakage at the thylakoid membrane, to (ii) preferentially bind there, and to (iii) seal leaky membrane regions via membrane fusion processes.

GTP hydrolysis by Synechocystis IM30 does not decisively affect its membrane remodeling activity.

The function of IM30 (also known as Vipp1) is linked to protection and/or remodeling of the thylakoid membrane system in chloroplasts and cyanobacteria. Recently, it has been revealed that the Arabidopsis IM30 protein exhibits GTP hydrolyzing activity in vitro, which was unexpected, as IM30 does not show any classical GTPase features. In the present study, we addressed the question, whether an apparent GTPase activity is conserved in IM30 proteins and can also be observed for IM30 of the cyanobacterium Synechocystis sp. PCC 6803. We show that Synechocystis IM30 is indeed able to bind and hydrolyze GTP followed by the release of Pi. Yet, the apparent GTPase activity of Synechocystis IM30 does not depend on Mg2+, which, together with the lack of classical GTPase features, renders IM30 an atypical GTPase. To elucidate the impact of this cryptic GTPase activity on the membrane remodeling activity of IM30, we tested whether GTP hydrolysis influences IM30 membrane binding and/or IM30-mediated membrane fusion. We show that membrane remodeling by Synechocystis IM30 is slightly affected by nucleotides. Yet, despite IM30 clearly catalyzing GTP hydrolysis, this does not seem to be vital for its membrane remodeling function.

Functional Implications of Multiple IM30 Oligomeric States.

The inner membrane-associated protein of 30 kDa (IM30), also known as the vesicle-inducing protein in plastids 1 (Vipp1), is essential for photo-autotrophic growth of cyanobacteria, algae and higher plants. While its exact function still remains largely elusive, it is commonly accepted that IM30 is crucially involved in thylakoid membrane biogenesis, stabilization and/or maintenance. A characteristic feature of IM30 is its intrinsic propensity to form large homo-oligomeric protein complexes. 15 years ago, it has been reported that these supercomplexes have a ring-shaped structure. However, the in vivo significance of these ring structures is not finally resolved yet and the formation of more complex assemblies has been reported. We here present and discuss research on IM30 conducted within the past 25 years with a special emphasis on the question of why we potentially need IM30 supercomplexes in vivo.

Mg2+ binding triggers rearrangement of the IM30 ring structure, resulting in augmented exposure of hydrophobic surfaces competent for membrane binding.

The "inner membrane-associated protein of 30 kDa" (IM30), also known as "vesicle-inducing protein in plastids 1" (Vipp1), is found in the majority of photosynthetic organisms that use oxygen as an energy source, and its occurrence appears to be coupled to the existence of thylakoid membranes in cyanobacteria and chloroplasts. IM30 is most likely involved in thylakoid membrane biogenesis and/or maintenance, and has recently been shown to function as a membrane fusion protein in presence of Mg2+ However, the precise role of Mg2+ in this process and its impact on the structure and function of IM30 remains unknown. Here, we show that Mg2+ binds directly to IM30 with a binding affinity of ∼1 mm Mg2+ binding compacts the IM30 structure coupled with an increase in the thermodynamic stability of the proteins' secondary, tertiary, and quaternary structures. Furthermore, the structural alterations trigger IM30 double ring formation in vitro because of increased exposure of hydrophobic surface regions. However, in vivo Mg2+-triggered exposure of hydrophobic surface regions most likely modulates membrane binding and induces membrane fusion.

What is Vipp1 good for?

While Vipp1 (also known as IM30) clearly is essential for proper biogenesis of thylakoid membranes in chloroplasts and cyanobacteria, the exact function of Vipp1/IM30 still remains unclear. The recent in vivo study of Gutu et al. now demonstrates that Vipp1/IM30 forms localized puncta specifically at highly curved membrane regions at the cell periphery. These Vipp1/IM30 puncta were found being highly dynamic under normal growth conditions, while it has recently been shown that they stably associate with membranes under high-light conditions. These observations, together with the observation that other Vipp1/IM30 homologous proteins also form puncta under stress conditions, indicate a protective function of these proteins at stressed membrane regions. However, Gutu et al. additionally show that Vipp1/IM30 is of special importance when growing cells are shifted from dark to light growth conditions, which could be explained by light stress, by thylakoid membrane dynamics and/or by photosystem biogenesis, which is being discussed in this article.