Bacterial Vipp1 and PspA are members of the ancient ESCRT-III membrane-remodeling superfamily.

Membrane remodeling and repair are essential for all cells. Proteins that perform these functions include Vipp1/IM30 in photosynthetic plastids, PspA in bacteria, and ESCRT-III in eukaryotes. Here, using a combination of evolutionary and structural analyses, we show that these protein families are homologous and share a common ancient evolutionary origin that likely predates the last universal common ancestor. This homology is evident in cryo-electron microscopy structures of Vipp1 rings from the cyanobacterium Nostoc punctiforme presented over a range of symmetries. Each ring is assembled from rungs that stack and progressively tilt to form dome-shaped curvature. Assembly is facilitated by hinges in the Vipp1 monomer, similar to those in ESCRT-III proteins, which allow the formation of flexible polymers. Rings have an inner lumen that is able to bind and deform membranes. Collectively, these data suggest conserved mechanistic principles that underlie Vipp1, PspA, and ESCRT-III-dependent membrane remodeling across all domains of life.

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.

Structure of the post-translational protein translocation machinery of the ER membrane.

Many proteins must translocate through the protein-conducting Sec61 channel in the eukaryotic endoplasmic reticulum membrane or the SecY channel in the prokaryotic plasma membrane1,2. Proteins with highly hydrophobic signal sequences are first recognized by the signal recognition particle (SRP)3,4 and then moved co-translationally through the Sec61 or SecY channel by the associated translating ribosome. Substrates with less hydrophobic signal sequences bypass the SRP and are moved through the channel post-translationally5,6. In eukaryotic cells, post-translational translocation is mediated by the association of the Sec61 channel with another membrane protein complex, the Sec62-Sec63 complex7-9, and substrates are moved through the channel by the luminal BiP ATPase9. How the Sec62-Sec63 complex activates the Sec61 channel for post-translational translocation is not known. Here we report the electron cryo-microscopy structure of the Sec complex from Saccharomyces cerevisiae, consisting of the Sec61 channel and the Sec62, Sec63, Sec71 and Sec72 proteins. Sec63 causes wide opening of the lateral gate of the Sec61 channel, priming it for the passage of low-hydrophobicity signal sequences into the lipid phase, without displacing the channel's plug domain. Lateral channel opening is triggered by Sec63 interacting both with cytosolic loops in the C-terminal half of Sec61 and transmembrane segments in the N-terminal half of the Sec61 channel. The cytosolic Brl domain of Sec63 blocks ribosome binding to the channel and recruits Sec71 and Sec72, positioning them for the capture of polypeptides associated with cytosolic Hsp7010. Our structure shows how the Sec61 channel is activated for post-translational protein translocation.

PDI family protein ERp29 recognizes P-domain of molecular chaperone calnexin.

Endoplasmic reticulum (ER) resident lectin chaperone calnexin (CNX) and calreticulin (CRT) assist folding of nascent glycoproteins. Their association with ERp57, a member of PDI family proteins (PDIs) which promote disulfide bond formation of unfolded proteins, has been well documented. Recent studies have provided evidence that other PDIs may also interact with CNX and CRT. Accordingly, it seems possible that the ER provides a repertoire of CNX/CRT-PDI complexes, in order to facilitate refolding of various glycoproteins. In this study, we examined the ability of PDIs to interact with CNX. Among them ERp29 was shown to interact with CNX, similarly to ERp57. Judging from the dissociation constant, its ability to interact with CNX was similar to that of ERp57. Results of further analyses by using a CNX mutant imply that ERp29 and ERp57 recognize the same domain of CNX, whereas the mode of interaction with CNX might be somewhat different between them.

Human GRP78 affinity towards its signaling partners Ire1α and PERK is differently modulated by an unfolded protein client.

Protein-folding stress is characteristic of specialized secretory cells and plays a dominant role in a multitude of diseases. The unfolded protein response (UPR) thus triggered is a proteostatic signaling network that adapts the protein-folding capacity of the endoplasmic reticulum to the cellular demands. We have measured the binding affinities between human GRP78, an essential chaperone located in ER, and two transmembrane UPR sensors (human PERK and Ire1α), with or without the addition of an unfolded protein client. We reveal distinct binding affinities between the binary and ternary complexes thus formed, that suggest a preference for the PERK signaling branch under stress, and a predilection for the GRP78-UPR sensor complex formation upon stressor removal. These results imply a gated UPR mechanism that tunes the overall cellular behavior to the accumulation of unfolded proteins.

Motor mechanism for protein threading through Hsp104.

The protein-remodeling machine Hsp104 dissolves amorphous aggregates as well as ordered amyloid assemblies such as yeast prions. Force generation originates from a tandem AAA+ (ATPases associated with various cellular activities) cassette, but the mechanism and allostery of this action remain to be established. Our cryoelectron microscopy maps of Hsp104 hexamers reveal substantial domain movements upon ATP binding and hydrolysis in the first nucleotide-binding domain (NBD1). Fitting atomic models of Hsp104 domains to the EM density maps plus supporting biochemical measurements show how the domain movements displace sites bearing the substrate-binding tyrosine loops. This provides the structural basis for N- to C-terminal substrate threading through the central cavity, enabling a clockwise handover of substrate in the NBD1 ring and coordinated substrate binding between NBD1 and NBD2. Asymmetric reconstructions of Hsp104 in the presence of ATPgammaS or ATP support sequential rather than concerted ATP hydrolysis in the NBD1 ring.

Low sequence identity but high structural and functional conservation: The case of Hsp70/Hsp90 organizing protein (Hop/Sti1) of Leishmania braziliensis.

Parasites belonging to the genus Leishmania are subjected to extensive environmental changes during their life cycle; molecular chaperones/co-chaperones act as protagonists in this scenario to maintain cellular homeostasis. Hop/Sti1 is a co-chaperone that connects the Hsp90 and Hsp70 systems, modulating their ATPase activities and affecting the fate of client proteins because it facilitates their transfer from the Hsp70 to the Hsp90 chaperone. Hop/Sti1 is one of the most prevalent co-chaperones, highlighting its importance despite the relatively low sequence identity among orthologue proteins. This multi-domain protein comprises three tetratricopeptides domains (TPR1, TPR2A and TPR2B) and two Asp/Pro-rich domains. Given the importance of Hop/Sti1 for the chaperone system and for Leishmania protozoa viability, the Leishmania braziliensis Hop (LbHop) and a truncated mutant (LbHop(TPR2AB)) were characterized. Structurally, both proteins are α-helix-rich and highly elongated monomeric proteins. Functionally, they inhibited the ATPase activity of Leishmania braziliensis Hsp90 (LbHsp90) to a similar extent, and the thermodynamic parameters of their interactions with LbHsp90 were similar, indicating that TPR2A-TPR2B forms the functional center for the LbHop interaction with LbHsp90. These results highlight the structural and functional similarity of Hop/Sti1 proteins, despite their low sequence conservation compared to the Hsp70 and Hsp90 systems, which are phylogenetic highly conserved.

Directly Observing the Lipid-Dependent Self-Assembly and Pore-Forming Mechanism of the Cytolytic Toxin Listeriolysin O.

Listeriolysin O (LLO) is the major virulence factor of Listeria monocytogenes and a member of the cholesterol-dependent cytolysin (CDC) family. Gram-positive pathogenic bacteria produce water-soluble CDC monomers that bind cholesterol-dependent to the lipid membrane of the attacked cell or of the phagosome, oligomerize into prepores, and insert into the membrane to form transmembrane pores. However, the mechanisms guiding LLO toward pore formation are poorly understood. Using electron microscopy and time-lapse atomic force microscopy, we show that wild-type LLO binds to membranes, depending on the presence of cholesterol and other lipids. LLO oligomerizes into arc- or slit-shaped assemblies, which merge into complete rings. All three oligomeric assemblies can form transmembrane pores, and their efficiency to form pores depends on the cholesterol and the phospholipid composition of the membrane. Furthermore, the dynamic fusion of arcs, slits, and rings into larger rings and their formation of transmembrane pores does not involve a height difference between prepore and pore. Our results reveal new insights into the pore-forming mechanism and introduce a dynamic model of pore formation by LLO and other CDC pore-forming toxins.

Structural dynamics of the MecA-ClpC complex: a type II AAA+ protein unfolding machine.

The MecA-ClpC complex is a bacterial type II AAA(+) molecular machine responsible for regulated unfolding of substrates, such as transcription factors ComK and ComS, and targeting them to ClpP for degradation. The six subunits of the MecA-ClpC complex form a closed barrel-like structure, featured with three stacked rings and a hollow passage, where substrates are threaded and translocated through successive pores. Although the general concepts of how polypeptides are unfolded and translocated by internal pore loops of AAA(+) proteins have long been conceived, the detailed mechanistic model remains elusive. With cryoelectron microscopy, we captured four different structures of the MecA-ClpC complexes. These complexes differ in the nucleotide binding states of the two AAA(+) rings and therefore might presumably reflect distinctive, representative snapshots from a dynamic unfolding cycle of this hexameric complex. Structural analysis reveals that nucleotide binding and hydrolysis modulate the hexameric complex in a number of ways, including the opening of the N-terminal ring, the axial and radial positions of pore loops, the compactness of the C-terminal ring, as well as the relative rotation between the two nucleotide-binding domain rings. More importantly, our structural and biochemical data indicate there is an active allosteric communication between the two AAA(+) rings and suggest that concerted actions of the two AAA(+) rings are required for the efficiency of the substrate unfolding and translocation. These findings provide important mechanistic insights into the dynamic cycle of the MecA-ClpC unfoldase and especially lay a foundation toward the complete understanding of the structural dynamics of the general type II AAA(+) hexamers.

Structural basis for the regulated protease and chaperone function of DegP.

All organisms have to monitor the folding state of cellular proteins precisely. The heat-shock protein DegP is a protein quality control factor in the bacterial envelope that is involved in eliminating misfolded proteins and in the biogenesis of outer-membrane proteins. Here we describe the molecular mechanisms underlying the regulated protease and chaperone function of DegP from Escherichia coli. We show that binding of misfolded proteins transforms hexameric DegP into large, catalytically active 12-meric and 24-meric multimers. A structural analysis of these particles revealed that DegP represents a protein packaging device whose central compartment is adaptable to the size and concentration of substrate. Moreover, the inner cavity serves antagonistic functions. Whereas the encapsulation of folded protomers of outer-membrane proteins is protective and might allow safe transit through the periplasm, misfolded proteins are eliminated in the molecular reaction chamber. Oligomer reassembly and concomitant activation on substrate binding may also be critical in regulating other HtrA proteases implicated in protein-folding diseases.

Bilberry anthocyanins neutralize the cytotoxicity of co-chaperonin GroES fibrillation intermediates.

The co-chaperonin GroES (Hsp10) works with chaperonin GroEL (Hsp60) to facilitate the folding reactions of various substrate proteins. Upon forming a specific disordered state in guanidine hydrochloride, GroES is able to self-assemble into amyloid fibrils similar to those observed in various neurodegenerative diseases. GroES therefore is a suitable model system to understand the mechanism of amyloid fibril formation. Here, we determined the cytotoxicity of intermediate GroES species formed during fibrillation. We found that neuronal cell death was provoked by soluble intermediate aggregates of GroES, rather than mature fibrils. The data suggest that amyloid fibril formation and its associated toxicity toward cell might be an inherent property of proteins irrespective of their correlation with specific diseases. Furthermore, with the presence of anthocyanins that are abundant in bilberry, we could inhibit both fibril formation and the toxicity of intermediates. Addition of bilberry anthocyanins dissolved the toxic intermediates and fibrils, and the toxicity of the intermediates was thus neutralized. Our results suggest that anthocyanins may display a general and potent inhibitory effect on the amyloid fibril formation of various conformational disease-causing proteins.

CryoEM structure of Hsp104 and its mechanistic implication for protein disaggregation.

Hsp104 is a ring-forming AAA+ machine that recognizes both aggregated proteins and prion-fibrils as substrates and, together with the Hsp70 system, remodels substrates in an ATP-dependent manner. Whereas the ability to disaggregate proteins is dependent on the Hsp104 M-domain, the location of the M-domain is controversial and its exact function remains unknown. Here we present cryoEM structures of two Hsp104 variants in both crosslinked and noncrosslinked form, in addition to the structure of a functional Hsp104 chimera harboring T4 lysozyme within the M-domain helix L2. Unexpectedly, we found that our Hsp104 chimera has gained function and can solubilize heat-aggregated beta-galactosidase (beta-gal) in the absence of the Hsp70 system. Our fitted structures confirm that the subunit arrangement of Hsp104 is similar to other AAA+ machines, and place the M-domains on the Hsp104 exterior, where they can potentially interact with large, aggregated proteins.

A missense mutation in the alphaB-crystallin chaperone gene causes a desmin-related myopathy.

Desmin-related myopathies (DRM) are inherited neuromuscular disorders characterized by adult onset and delayed accumulation of aggregates of desmin, a protein belonging to the type III intermediate filament family, in the sarcoplasma of skeletal and cardiac muscles. In this paper, we have mapped the locus for DRM in a large French pedigree to a 26-cM interval in chromosome 11q21-23. This region contains the alphaB-crystallin gene (CRYAB), a candidate gene encoding a 20-kD protein that is abundant in lens and is also present in a number of non-ocular tissues, including cardiac and skeletal muscle. AlphaB-crystallin is a member of the small heat shock protein (shsp) family and possesses molecular chaperone activity. We identified an R120G missense mutation in CRYAB that co-segregates with the disease phenotype in this family. Muscle cell lines transfected with the mutant CRYAB cDNA showed intracellular aggregates that contain both desmin and alphaB-crystallin as observed in muscle fibers from DRM patients. These results are the first to identify a defect in a molecular chaperone as a cause for an inherited human muscle disorder.

Aggregate reactivation mediated by the Hsp100 chaperones.

Hsp100 family of molecular chaperones shows a unique capability to resolubilize and reactivate aggregated proteins. The Hsp100-mediated protein disaggregation is linked to the activity of other chaperones from the Hsp70 and Hsp40 families. The best-studied members of the Hsp100 family are the bacterial ClpB and Hsp104 from yeast. Hsp100 chaperones are members of a large super-family of energy-driven conformational "machines" known as AAA+ ATPases. This review describes the current mechanistic model of the chaperone-induced protein disaggregation and explains how the structural architecture of Hsp100 supports disaggregation and how the co-chaperones may participate in the Hsp100-mediated reactions.

Bowl-shaped oligomeric structures on membranes as DegP's new functional forms in protein quality control.

In the periplasm of Escherichia coli, DegP (also known as HtrA), which has both chaperone-like and proteolytic activities, prevents the accumulation of toxic misfolded and unfolded polypeptides. In solution, upon binding to denatured proteins, DegP forms large cage-like structures. Here, we show that DegP forms a range of bowl-shaped structures, independent of substrate proteins, each with a 4-, 5-, or 6-fold symmetry and all with a DegP trimer as the structural unit, on lipid membranes. These membrane-bound DegP assemblies have the capacity to recruit and process substrates in the bowl chamber, and they exhibit higher proteolytic and lower chaperone-like activities than DegP in solution. Our findings imply that DegP might regulate its dual roles during protein quality control, depending on its assembly state in the narrow bacterial envelope.

Interaction of constituents of MDT regimen for leprosy with Mycobacterium leprae HSP18: impact on its structure and function.

Mycobacterium leprae, the causative organism of leprosy, harbors many antigenic proteins, and one such protein is the 18-kDa antigen. This protein belongs to the small heat shock protein family and is commonly known as HSP18. Its chaperone function plays an important role in the growth and survival of M. leprae inside infected hosts. HSP18/18-kDa antigen is often used as a diagnostic marker for determining the efficacy of multidrug therapy (MDT) in leprosy. However, whether MDT drugs (dapsone, clofazimine, and rifampicin) do interact with HSP18 and how these interactions affect its structure and chaperone function is still unclear. Here, we report evidence of HSP18-dapsone/clofazimine/rifampicin interaction and its impact on the structure and chaperone function of HSP18. These three drugs interact efficiently with HSP18 (having submicromolar binding affinity) with 1 : 1 stoichiometry. Binding of these MDT drugs to the 'α-crystallin domain' of HSP18 alters its secondary structure and tryptophan micro-environment. Furthermore, surface hydrophobicity, oligomeric size, and thermostability of the protein are reduced upon interaction with these three drugs. Eventually, all these structural alterations synergistically decrease the chaperone function of HSP18. Interestingly, the effect of rifampicin on the structure, stability, and chaperone function of this mycobacterial small heat shock protein is more pronounced than the other two MDT drugs. This reduction in the chaperone function of HSP18 may additionally abate M. leprae survivability during multidrug treatment. Altogether, this study provides a possible foundation for rational designing and development of suitable HSP18 inhibitors in the context of effective treatment of leprosy.

Activation of DegP chaperone-protease via formation of large cage-like oligomers upon binding to substrate proteins.

Cells use molecular chaperones and proteases to implement the essential quality control mechanism of proteins. The DegP (HtrA) protein, essential for the survival of Escherichia coli cells at elevated temperatures with homologues found in almost all organisms uniquely has both functions. Here we report a mechanism for DegP to activate both functions via formation of large cage-like 12- and 24-mers after binding to substrate proteins. Cryo-electron microscopic and biochemical studies revealed that both oligomers are consistently assembled by blocks of DegP trimers, via pairwise PDZ1-PDZ2 interactions between neighboring trimers. Such interactions simultaneously eliminate the inhibitory effects of the PDZ2 domain. Additionally, both DegP oligomers were also observed in extracts of E. coli cells, strongly implicating their physiological importance.

Cryo electron microscopy structures of Hsp100 proteins: crowbars in or out?

Independent cryo electron microscopy (cryo-EM) studies of the closely related protein disaggregases ClpB and Hsp104 have resulted in two different models of subunit arrangement in the active hexamer. We compare the EM maps and resulting atomic structure fits, discuss their differences, and relate them to published experimental information in an attempt to discriminate between models. In addition, we present some general assessment criteria for low-resolution cryo-EM maps to offer non-structural biologists tools to evaluate these structures.

GroEL assisted folding of large polypeptide substrates in Escherichia coli: Present scenario and assignments for the future.

Escherichia coli chaperonins GroEL and GroES are indispensable for survival and growth of the cell since they provide essential assistance to the folding of many newly translated proteins in the cell. Recent studies indicate that a substantial portion of the proteins involved in the host pathways are completely dependent on GroEL-GroES for their folding and hence providing some explanation for why GroEL is essential for cell growth. Many proteins either small-single domain or large multidomains require assistance from GroEL-ES during their lifetime. Proteins of size up to approximately 70kDa can fold via the cis mechanism during GroEL-ES assisted pathway, but other proteins (>70kDa) that cannot be pushed inside the cavity of GroEL-ATP complex upon binding of GroES fold by an evolved mechanism called trans. In recent years, much work has been done on revealing facts about the cis mechanism involving the GroEL assisted folding of small proteins whereas the trans mechanism with larger polypeptide substrates still remains under cover. In order to disentangle the role of chaperonin GroEL-GroES in the folding of large E. coli proteins, this review discusses a number of issues like the range of large polypeptide substrates acted on by GroEL. Do all these substrates need the complete chaperonin system along with ATP for their folding? Does GroEL act as foldase or holdase during the process? We conclude with a discussion of the various queries that need to be resolved in the future for an extensive understanding of the mechanism of GroEL mediated folding of large substrate proteins in E. coli cytosol.

A library of protein cage architectures as nanomaterials.

Virus capsids and other structurally related cage-like proteins such as ferritins, dps, and heat shock proteins have three distinct surfaces (inside, outside, interface) that can be exploited to generate nanomaterials with multiple functionality by design. Protein cages are biological in origin and each cage exhibits extremely homogeneous size distribution. This homogeneity can be used to attain a high degree of homogeneity of the templated material and its associated property. A series of protein cages exhibiting diversity in size, functionality, and chemical and thermal stabilities can be utilized for materials synthesis under a variety of conditions. Since synthetic approaches to materials science often use harsh temperature and pH, it is an advantage to utilize protein cages from extreme environments. In this chapter, we review recent studies on discovering novel protein cages from harsh natural environments such as the acidic thermal hot springs at Yellowstone National Park (YNP) and on utilizing protein cages as nano-scale platforms for developing nanomaterials with wide range of applications from electronics to biomedicine.