Jeremy H. Lakey

Exome sequencing identifies GATA-2 mutation as the cause of dendritic cell, monocyte, B and NK lymphoid deficiency

The human syndrome of dendritic cell, monocyte, B and natural killer lymphoid deficiency presents as a sporadic or autosomal dominant trait causing susceptibility to mycobacterial and other infections, predisposition to myelodysplasia and leukemia, and, in some cases, pulmonary alveolar proteinosis. Seeking a genetic cause, we sequenced the exomes of 4 unrelated persons, 3 with sporadic disease, looking for novel, heterozygous, and probably deleterious variants. A number of genes harbored novel variants in person, but only one gene, GATA2, was mutated in all 4 persons. Each person harbored a different mutation, but all were predicted to be highly deleterious and to cause loss or mutation of the C-terminal zinc finger domain. Because GATA2 is the only common mutated gene in 4 unrelated persons, it is highly probable to be the cause of dendritic cell, monocyte, B, and natural killer lymphoid deficiency. This disorder therefore constitutes a new genetic form of heritable immunodeficiency and leukemic transformation.

The bacterial porin superfamily: sequence alignment and structure prediction

The porins of Gram‐negative bacteria are responsible for the ‘molecular sieve’ properties of the outer membrane. They form large water‐filled channels which allow the diffusion of hydrophilic molecules into the periplasmic space. Owing to the strong hydrophilicity of their amino acid sequence and the nature of their secondary structure (beta strands), conventional hydropathy methods for predicting membrane topology are useless for this class of protein. The large number of available porin amino acid sequences was exploited to improve the accuracy of the prediction In combination with tools detecting amphipathicity of secondary structure. Using the constraints of β‐sheet structure these porins are predicted to contain 16 membrane‐spanning strands, 14 of which are common to the two (enteric and the neisserial) porin subfamilies.

Heat does not come in different colours: entropy–enthalpy compensation, free energy windows, quantum confinement, pressure perturbation calorimetry, solvation and the multiple causes of heat capacity effects in biomolecular interactions

Modern techniques in microcalorimetry allow us to measure directly the heat changes and associated thermodynamics for biomolecular processes in aqueous solution at reasonable concentrations. All these processes involve changes in solvation/hydration, and it is natural to assume that the heats for these processes should reflect, in some way, such changes in solvation. However, the interpretation of data is still somewhat ambiguous, since different non-covalent interactions may have similar thermodynamic signatures, and analysis is frustrated by large entropy-enthalpy compensation effects. Changes in heat capacity (Delta C(p)) have been related to changes in hydrophobic hydration and non-polar accessible surface areas, but more recent empirical and theoretical work has shown how this need not always be the case. Entropy-enthalpy compensation is a natural consequence of finite Delta C(p) values and, more generally, can arise as a result of quantum confinement effects, multiple weak interactions, and limited free energy windows, giving rise to thermodynamic homeostasis that may be of evolutionary and functional advantage. The new technique of pressure perturbation calorimetry (PPC) has enormous potential here as a means of probing solvation-related volumetric changes in biomolecules at modest pressures, as illustrated with preliminary data for a simple protein-inhibitor complex.

Two-step Membrane Binding by Equinatoxin II, a Pore-forming Toxin from the Sea Anemone, Involves an Exposed Aromatic Cluster and a Flexible Helix

Equinatoxin II (EqtII) belongs to a unique family of 20-kDa pore-forming toxins from sea anemones. These toxins preferentially bind to membranes containing sphingomyelin and create cation-selective pores by oligomerization of 3–4 monomers. In this work we have studied the binding of EqtII to lipid membranes by the use of lipid monolayers and surface plasmon resonance (SPR). The binding is a two-step process, separately mediated by two regions of the molecule. An exposed aromatic cluster involving tryptophans 112 and 116 mediates the initial attachment that is prerequisite for the next step. Steric shielding of the aromatic cluster or mutation of Trp-112 and -116 to phenylalanine significantly reduces the toxin-lipid interaction. The second step is promoted by the N-terminal amphiphilic helix, which translocates into the lipid phase. The two steps were distinguished by the use of a double cysteine mutant having the N-terminal helix fixed to the protein core by a disulfide bond. The kinetics of membrane binding derived from the SPR experiments could be fitted to a two-stage binding model. Finally, by using membrane-embedded quenchers, we showed that EqtII does not insert deeply in the membrane. The first step of the EqtII binding is reminiscent of the binding of the evolutionarily distant cholesterol-dependant cytolysins, which share a similar structural motif in the membrane attachment domain.

Amphipols: Polymeric surfactants for membrane biology research

Membrane proteins classically are handled in aqueous solutions as complexes with detergents. The dissociating character of detergents, combined with the need to maintain an excess of them, frequently results in more or less rapid inactivation of the protein under study. Over the past few years, we have endeavored to develop a novel family of surfactants, dubbed amphipols (APs). APs are amphiphilic polymers that bind to the transmembrane surface of the protein in a noncovalent but, in the absence of a competing surfactant, quasi-irreversible manner. Membrane proteins complexed by APs are in their native state, stable, and they remain water-soluble in the absence of detergent or free APs. An update is presented of the current knowledge about these compounds and their demonstrated or putative uses in membrane biology.

Molecular Determinants of Sphingomyelin Specificity of a Eukaryotic Pore-forming Toxin

Sphingomyelin (SM) is abundant in the outer leaflet of the cell plasma membrane, with the ability to concentrate in so-called lipid rafts. These specialized cholesterol-rich microdomains not only are associated with many physiological processes but also are exploited as cell entry points by pathogens and protein toxins. SM binding is thus a widespread and important biochemical function, and here we reveal the molecular basis of SM recognition by the membrane-binding eukaryotic cytolysin equinatoxin II (EqtII). The presence of SM in membranes drastically improves the binding and permeabilizing activity of EqtII. Direct binding assays showed that EqtII specifically binds SM, but not other lipids and, curiously, not even phosphatidylcholine, which presents the same phosphorylcholine headgroup. Analysis of the EqtII interfacial binding site predicts that electrostatic interactions do not play an important role in the membrane interaction and that the two most important residues for sphingomyelin recognition are Trp112 and Tyr113 exposed on a large loop. Experiments using site-directed mutagenesis, surface plasmon resonance, lipid monolayer, and liposome permeabilization assays clearly showed that the discrimination between sphingomyelin and phosphatidylcholine occurs in the region directly below the phosphorylcholine headgroup. Because the characteristic features of SM chemistry lie in this subinterfacial region, the recognition mechanism may be generic for all SM-specific proteins.

Crystal structure of a colicin N fragment suggests a model for toxicity

BACKGROUND Pore-forming colicins are water-soluble bacteriocins capable of binding to and translocating through the Escherichia coli cell envelope. They then undergo a transition to a transmembrane ion channel in the cytoplasmic membrane leading to bacterial death. Colicin N is the smallest pore-forming colicin known to date (40 kDa instead of the more usual 60 kDa) and the crystal structure of its membrane receptor, the porin OmpF, is already known. Structural knowledge of colicin N is therefore important for a molecular understanding of colicin toxicity and is relevant to toxic mechanisms in general. RESULTS The crystal structure of colicin N reveals a novel receptor-binding domain containing a six-stranded antiparallel beta sheet wrapped around the 63 A long N-terminal alpha helix of the pore-forming domain. The pore-forming domain adopts a ten alpha-helix bundle that has been observed previously in the pore-forming domains of colicin A, la and E1. The translocation domain, however, does not appear to adopt any regular structure. Models for receptor binding and translocation through the outer membrane are proposed based on the structure and biochemical data. CONCLUSIONS The colicin N-ompF system is now the structurally best-defined translocation pathway. Knowledge of the colicin N structure, coupled with the structure of its receptor, OmpF, and previously published biochemical data, limits the numerous possibilities of translocation and leads to a model in which the translocation domain inserts itself through the porin pore, the receptor-binding domain stays outside and the pore-forming domain translocates along the outer wall of the trimeric porin channel.

Disparate proteins use similar architectures to damage membranes

Membrane disruption can efficiently alter cellular function; indeed, pore-forming toxins (PFTs) are well known as important bacterial virulence factors. However, recent data have revealed that structures similar to those found in PFTs are found in membrane active proteins across disparate phyla. Many similarities can be identified only at the 3D-structural level. Of note, domains found in membrane-attack complex proteins of complement and perforin (MACPF) resemble cholesterol-dependent cytolysins from Gram-positive bacteria, and the Bcl family of apoptosis regulators share similar architectures with Escherichia coli pore-forming colicins. These and other correlations provide considerable help in understanding the structural requirements for membrane binding and pore formation.

Pore-forming colicins and their relatives

The pore-forming colicins, the first proteins that were capable of forming voltage-dependent ion channels to be sequenced, have turned out to be both less tractable and more mysterious than imagined; yet they have proved interesting at every step of their short journey from producing cell to vanquished target cell. Starting out as a remarkably extended water-soluble protein, the colicin molecule is designed to interact simultaneously with several components of the complex membrane of the target cell, transform itself into a membrane protein, and become an ion channel with inscrutable properties. Unraveling how it does all this appears to be leading us into the dark recesses of protein/protein and protein/membrane interaction, where lurk fundamental processes reluctantly waiting to be revealed.

THE MECHANISM OF PHOSPHORYLATION-INDUCIBLE ACTIVATION OF THE ETS-DOMAIN TRANSCRIPTION FACTOR ELK-1

Protein phosphorylation represents one of the major mechanisms for transcription factor activation. Here we demonstrate a molecular mechanism by which phosphorylation by mitogen‐activated protein (MAP) kinases leads to changes in transcription factor activity. MAP kinases stimulate DNA binding and transcriptional activation mediated by the mammalian ETS‐domain transcription factor Elk‐1. Phosphorylation of the C‐terminal transcriptional activation domain induces a conformational change in Elk‐1, which accompanies the stimulation of DNA binding. C‐terminal phosphorylation is coupled to activation of DNA binding by the N‐terminal DNA‐binding domain via an additional intermediary domain. Activation of DNA binding is mediated by an allosteric mechanism involving the key phosphoacceptor residues. Together, these results provide a molecular model for how phosphorylation induces changes in Elk‐1 activity.