Pore-forming toxins

Pore-forming toxins are widely distributed proteins which form lesions in biological membranes. In this review, bacterial pore-forming toxins are treated as a paradigm and discussed in terms of the structural principles on which they work. Then, a large family of bacterial toxins, the cholesterol-binding toxins, are analyzed in depth to provide an overview of the processes involved in pore formation. The ways in which the cholesterol-binding toxins (cholesterol-dependent cytolysins) interact with membranes and form pores, the structure of the monomeric soluble and oligomeric pore-forming states, and the effects of the toxin on membrane structure are discussed. By surveying the range of work which has been done on cholesterol-binding toxins, a working model is elaborated which reconciles two current, apparently diametrically opposed, models for their mechanism. Received 7 September 2001; received after revision 12 November 2001; accepted 5 December 2001     

A molecular pin to study the dynamics of β-barrel formation in pore-forming toxins on erythrocytes: a sliding model

γ-Hemolysins are pore-forming toxins which develop from water-soluble monomers by combining two different ‘albeit homologous’ proteins. They form oligomeric pores in both cell and model membranes by undergoing a still poorly understood conformational rearrangement in the stem region. The stem is formed by three β-strands, folded onto the core of the soluble protein and completely extended in the pore. We propose a new model to explain such a process. Seven double-cysteine mutants were developed by inserting one cysteine on the stretch that links the β-hairpin to the core of the protein and another on different positions along the β-strands. The membrane bound protein was blocked in a non-lytic state by S–S bond formation. Six mutants were oxidized as inactive intermediates, but became active after adding DTT. These results demonstrate that the stem extension can be temporarily frozen and that the β-barrel formation occurs by β-strand concerted step-by-step sliding. Received 22 October 2007; received after revision 15 November 2007; accepted 19 November 2007     

Architecture and regulation of HtrA-family proteins involved in protein quality control and stress response

Protein quality control is vital for all living cells and sophisticated molecular mechanisms have evolved to prevent the excessive accumulation of unfolded proteins. High-temperature requirement A (HtrA) proteases have been identified as important ATP-independent quality-control factors in most species. HtrA proteins harbor a serine-protease domain and at least one peptide-binding PDZ domain to ensure efficient removal of misfolded or damaged proteins. One distinctive property of HtrAs is their ability to assemble into complex oligomers. Whereas all examined HtrAs are capable of forming pyramidal 3-mers, higher-order complexes consisting of up to 24 molecules have been reported. Tight control of chaperone and protease function is of pivotal importance in preventing deleterious HtrA-protease activity. In recent years, structural biology provided detailed insights into the molecular basis of the regulatory mechanisms, which include unique intramolecular allosteric signaling cascades and the dynamic switching of oligomeric states of HtrA proteins. Based on these results, functional models for many family members have been developed. The HtrA protein family represents a remarkable example of how structural and functional diversity is attained from the assembly of simple molecular building blocks.     

Transformation of amyloid β(1–40) oligomers into fibrils is characterized by a major change in secondary structure

Alzheimer’s disease (AD) is a neurodegenerative disorder occurring in the elderly. It is widely accepted that the amyloid beta peptide (Aβ) aggregation and especially the oligomeric states rather than fibrils are involved in AD onset. We used infrared spectroscopy to provide structural information on the entire aggregation pathway of Aβ(1–40), starting from monomeric Aβ to the end of the process, fibrils. Our structural study suggests that conversion of oligomers into fibrils results from a transition from antiparallel to parallel β-sheet. These structural changes are described in terms of H-bonding rupture/formation, β-strands reorientation and β-sheet elongation. As antiparallel β-sheet structure is also observed for other amyloidogenic proteins forming oligomers, reorganization of the β-sheet implicating a reorientation of β-strands could be a generic mechanism determining the kinetics of protein misfolding. Elucidation of the process driving aggregation, including structural transitions, could be essential in a search for therapies inhibiting aggregation or disrupting aggregates.     

An outline of desensitization in pentameric ligand-gated ion channel receptors

Pentameric ligand-gated ion channel (pLGIC) receptors exhibit desensitization, the progressive reduction in ionic flux in the prolonged presence of agonist. Despite its pathophysiological importance and the fact that it was first described over half a century ago, surprisingly little is known about the structural basis of desensitization in this receptor family. Here, we explain how desensitization is defined using functional criteria. We then review recent progress into reconciling the structural and functional basis of this phenomenon. The extracellular–transmembrane domain interface is a key locus. Activation is well known to involve conformational changes at this interface, and several lines of evidence suggest that desensitization involves a distinct conformational change here that is incompatible with activation. However, major questions remain unresolved, including the structural basis of the desensitization-induced agonist affinity increase and the mechanism of pore closure during desensitization.     

The utility F-box for protein destruction

A signature feature of all living organisms is their utilization of proteins to construct molecular machineries that undertake the complex network of cellular activities. The abundance of a protein element is temporally and spatially regulated in two opposing aspects: de novo synthesis to manufacture the required amount of the protein, and destruction of the protein when it is in excess or no longer needed. One major route of protein destruction is coordinated by a set of conserved molecules, the F-box proteins, which promote ubiquitination in the ubiquitin-proteasome pathway. Here we discuss the functions of F-box proteins in several cellular scenarios including cell cycle progression, synapse formation, plant hormone responses, and the circadian clock. We particularly emphasize the mechanisms whereby F-box proteins recruit specific substrates and regulate their abundance in the context of SCF E3 ligases. For some exceptions, we also review how F-box proteins function through non-SCF mechanisms.     

Structural and functional features of dimeric dihydrodiol dehydrogenase

Dimeric dihydrodiol dehydrogenase (DD) catalyzes the NADP(+)-dependent oxidation of trans-dihydrodiols of aromatic hydrocarbons to their corresponding catechols. The tertiary structure of dimeric DD consists of a classical dinucleotide binding domain comprising two betaalphabetaalphabeta motifs at the N-terminus, and an eight-stranded, predominantly anti-parallel beta-sheet, forming the C-terminal domain The aim of this review is to summarize the biochemical and structural properties of dimeric DD, compare it to enzymes that are structurally similar, and provide an insight into its catalytic mechanism and membership amongst a unique family of monomeric/oligomeric proteins that most likely share a common ancestry.     

Structure and function of interleukin-22 and other members of the interleukin-10 family

The IL-10 family of cytokines is comprised of IL-10, IL-19, IL-20, IL-22, IL-24, IL-26, and IFN-λs (IL-28A, IL-28B, and IL-29). The IL-10 family members bind to shared class II cytokine receptor chains that associate in various combinations in heterodimeric complexes. Upon interleukin/receptor complex formation, these proteins switch on the Jak/STAT pathway and elicit pleiotropic biological responses whose variety sharply contrasts with their structural similarities. IL-10 family members are involved in several human diseases and health conditions and hence their structural analyses may provide valuable information to design specific therapeutic strategies. In this review, we describe the human interleukin-10 family of cytokines, focusing on their structures and functions, with particular attention given to IL-22 and IL-10. We report on the recently published structures of IL-10 cytokine family members and their complexes with cognate transmembrane and soluble receptors as well as on interleukin physiology and physiopathology.     

The current structural and functional understanding of APOBEC deaminases

The apolipoprotein B mRNA-editing enzyme catalytic polypeptide (APOBEC) family of cytidine deaminases has emerged as an intensively studied field as a result of their important biological functions. These enzymes are involved in lipid metabolism, antibody diversification, and the inhibition of retrotransposons, retroviruses, and some DNA viruses. The APOBEC proteins function in these roles by deaminating single-stranded (ss) DNA or RNA. There are two high-resolution crystal structures available for the APOBEC family, Apo2 and the C-terminal catalytic domain (CD2) of Apo3G or Apo3G-CD2 [Holden et al. (Nature 456:121–124, 2008); Prochnow et al. (Nature 445:447–451, 2007)]. Additionally, the structure of Apo3G-CD2 has also been determined using NMR [Chen et al. (Nature 452:116–119, 2008); Furukawa et al. (EMBO J 28:440–451, 2009); Harjes et al. (J Mol Biol, 2009)]. A detailed structural analysis of the APOBEC proteins and a comparison to other zinc-coordinating deaminases can facilitate our understanding of how APOBEC proteins bind nucleic acids, recognize substrates, and form oligomers. Here, we review the recent development of structural and functional studies that apply to Apo3G as well as the APOBEC deaminase family.     

Many mechanisms, one entrance: membrane protein translocation into the nucleus

The inner nuclear membrane harbors a unique set of membrane proteins, many of which interact with nuclear intermediate filaments and chromatin components and thus play an important role in nuclear organization and gene expression regulation. These membrane proteins have to be constantly transported into the nucleus from their sites of synthesis in the ER to match the growth of the nuclear membrane during interphase. Many mechanisms have evolved to enable translocation of these proteins to the nucleus. The full range of mechanisms goes from rare autophagy events to regulated translocation using the nuclear pore complexes. Though mechanisms involving nuclear pores are predominant, within this group an enormous mechanistic range is observed from free diffusion through the peripheral channels to many distinct mechanisms involving different nucleoporins and other components of the soluble protein transport machinery in the central channels. This review aims to provide a comprehensive insight into this mechanistic diversity.     

Biogenesis of β-barrel membrane proteins in bacteria and eukaryotes: evolutionary conservation and divergence

Membrane-embedded β-barrel proteins span the membrane via multiple amphipathic β-strands arranged in a cylindrical shape. These proteins are found in the outer membranes of Gram-negative bacteria, mitochondria and chloroplasts. This situation is thought to reflect the evolutionary origin of mitochondria and chloroplasts from Gram-negative bacterial endosymbionts. β-barrel proteins fulfil a variety of functions; among them are pore-forming proteins that allow the flux of metabolites across the membrane by passive diffusion, active transporters of siderophores, enzymes, structural proteins, and proteins that mediate protein translocation across or insertion into membranes. The biogenesis process of these proteins combines evolutionary conservation of the central elements with some noticeable differences in signals and machineries. This review summarizes our current knowledge of the functions and biogenesis of this special family of proteins.     

Colicins: prokaryotic killer-pores.

Colicins are plasmid-encoded protein antibiotics which kill bacteria closely related to the producing strain (generally Escherichia coli). The study of the function of colicins has revealed many features which reflect common targeting and translocation mechanisms with bacteriophages and toxins. Like many toxins, colicins are composed of structural domains specialized in one of the different steps of the activity, targeting, translocation and killing. The major group comprises those colicins which permeabilize the cytoplasmic membrane, thereby destroying the cell's membrane potential. These colicins form well-defined voltage-gated ion channels in artificial membranes. The scope of this review is to describe some of the more recent findings concerning the structure and mode of action of pore-forming colicins with a special attention to models of membrane insertion and pore structure based on the recently determined three-dimensional structure of the pore-forming domain of colicin A.     

The role of chemokines and their receptors in angiogenesis

Chemokines are a vertebrate-specific group of small molecules that regulate cell migration and behaviour in diverse contexts. So far, around 50 chemokines have been identified in humans, which bind to 18 different chemokine receptors. These are members of the seven-transmembrane receptor family. Initially, chemokines were identified as modulators of the immune response. Subsequently, they were also shown to regulate cell migration during embryonic development. Here, we discuss the influence of chemokines and their receptors on angiogenesis, or the formation of new blood vessels. We highlight recent advances in our understanding of how chemokine signalling might directly influence endothelial cell migration. We furthermore examine the contributions of chemokine signalling in immune cells during this process. Finally, we explore possible implications for disease settings, such as chronic inflammation and tumour progression.     

Biophysical properties of porin pores from mitochondrial outer membrane of eukaryotic cells.

The matrix space of mitochondria is surrounded by two membranes. The mitochondrial inner membrane contains the respiration chain and a large number of highly specific carriers for the mostly anionic substrates of mitochondrial metabolism. In contrast to this the permeability properties of the mitochondrial outer membrane are by far less specific. It acts as a molecular sieve for hydrophilic molecules with a defined exclusion limit around 3000 Da. Responsible for the extremely high permeability of the mitochondrial outer membrane is the presence of a pore-forming protein termed mitochondrial porin. Mitochondrial porins have been isolated from a variety of eukaryotic cells. They are basic proteins with molecular masses between 30 and 35 kDa. Reconstitution experiments define their function as pore-forming components with a single-channel conductance of about 0.40 nS (nano Siemens) in 0.1 M KCl at low voltages. In the open state mitochondrial porin behaves as a general diffusion pore with an effective diameter of 1.7 nm. Eukaryotic porins are slightly anion-selective in the open state but become cation-selective after voltage-dependent closure.     

Biophysical properties of porin pores from mitochondrial outer membrane of eukaryotic cells

Summary The matrix space of mitochondria is surrounded by two membranes. The mitochondrial inner membrane contains the respiration chain and a large number of highly specific carriers for the mostly anionic substrates of mitochondrial metabolism. In contrast to this the permeability properties of the mitochondrial outer membrane are by far less specific. It acts as a molecular sieve for hydrophilic molecules with a defined exclusion limit around 3000 Da. Responsible for the extremely high permeability of the mitochondrial outer membrane is the presence of a pore-forming protein termed mitochondrial porin. Mitochondrial porins have been isolated from a variety of eukaryotic cells. They are basic proteins with molecular masses between 30 and 35 kDa. Reconstitution experiments define their function as pore-forming components with a single-channel conductance of about 0.40 nS (nano Siemens) in 0.1 M KCl at low voltages. In the open state mitochondrial porin behaves as a general diffusion pore with an effective diameter of 1.7 nm. Eukaryotic porins are slightly anion-selective in the open state but become cation-selective after voltage-dependent closure.     

Nuclear envelope and nuclear matrix: interactions and dynamics

The peripheral nuclear lamina is located near the nuclear inner membrane and consists of lamin filaments and integral membrane proteins, including the lamin B receptor and various isoforms of lamina-associated polypeptides (LAP) 1 and 2. Several nuclear membrane proteins also interact with chromatin proteins BAF and Hp1. Lamins in the nuclear interior associate with at least one soluble (non-membrane-bound) LAP2 isoform named LAP2alpha. The internal lamins, together with Tpr-based filaments that connect to nuclear pore complexes, are proposed to be major structural elements of the internal nuclear matrix. We describe the structural links between the peripheral lamina and the internal nuclear matrix that are thought to be mediated by LAP2 family members, filament protein Tpr and nucleoporin Nup153. These findings are discussed in relation to human diseases that arise from mutations in nuclear lamina proteins.     

Biological Roles of Neural J Proteins

J proteins are chief regulators of the Hsp70 family, a highly conserved family of ATPases that mediate conformational changes in a broad range of proteins. The J protein family has been the central focus of numerous prokaryote and eukaryote biologists. Common questions that arise include: How does the J protein/Hsp70 machinery support protein folding? What role do J proteins play in protein misfolding and neurodegenerative disorders? Can the J protein/ Hsp70 machinery be harnessed to provide a rational basis for recombinant protein production? The current progress that has resulted from the convergence of biochemistry with Escherichia coli and Saccharomyces cerevisiae genetics has accelerated the pace at which these questions are being elucidated. We are beginning to gain some insights into the neuronal network of J proteins. Here, we highlight recent advances in our understanding of how select J proteins harness Hsp70 s for fundamentally important conformational work in neurons.     

Molecular analysis of axonal target specificity and synapse formation

The development of neuronal connectivity requires the growth of axons to their target region and the formation of dendritic trees that extend into specific layers. Within the target region growth cones, the tips of extending axons are guided to finer target fields including specific subcellular compartments where they form synapses. In this article we highlight recent progress on molecular aspects of axonal subcellular target selection such as the axon initial segment or specific sublaminae of the vertebrate retina. We then discuss the very recent progress on the molecular analysis of synapse formation in the central nervous system, including the direction of differentiation into an inhibitory or excitatory synapse. Apparently, initial synaptic contacts are structurally and functionally modulated by neuronal activity, raising the question how neuronal activity can modify synaptic circuits. We therefore also focus on neural proteins that are up-regulated, secreted or converted by synaptic activity and, thus, might represent molecular candidates for experience-driven refinement or remodeling of synaptic connections. Received 5 July 2005; received after revision 19 August 2005; accepted 2 September 2005     

Structure-function relationships in methionine adenosyltransferases

Methionine adenosyltransferases (MATs) are the family of enzymes that synthesize the main biological methyl donor, S-adenosylmethionine. The high sequence conservation among catalytic subunits from bacteria and eukarya preserves key residues that control activity and oligomerization, which is reflected in the protein structure. However, structural differences among complexes with substrates and products have led to proposals of several reaction mechanisms. In parallel, folding studies begin to explain how the three intertwined domains of the catalytic subunit are produced, and to highlight the importance of certain intermediates in attaining the active final conformation. This review analyzes the available structural data and proposes a consensus interpretation that facilitates an understanding of the pathological problems derived from impairment of MAT function. In addition, new research opportunities directed toward clarification of aspects that remain obscure are also identified. Received 22 August 2008; received after revision 22 September 2008; accepted 26 September 2008