The 'molten' globular conformation of a protein is compact with a native secondary structure but a poorly defined tertiary structure. Molten globular states are intermediates in protein folding and unfolding and they may be involved in the translocation or insertion of proteins into membranes. Here we investigate the membrane insertion of the pore-forming domain of colicin A, a bacteriocin that depolarizes the cytoplasmic membrane of sensitive cells. We find that this pore-forming domain, the insertion of which depends on pH, undergoes a native to molten globule transition at acidic pH. The variation of the kinetic constant of membrane insertion of the protein into negatively charged lipid vesicles as a function of the interfacial pH correlates with the appearance of the acidic molten globular state, indicating that this state could be an intermediate formed during the insertion of colicin A into membranes.

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. (Blood. 2011;118(10):2656-2658)

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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 membraneembedded 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.Targeting and attachment of proteins to membranes is one of the key steps in many cellular processes (1-3). Protein-membrane interactions have been studied intensively in recent years with many different examples of proteins and membranes. These interactions can be promoted at the lipid-water interface by lipid anchors, electrostatic forces or surface-exposed aromatic and aliphatic residues (1, 2, 4). Compared with protein-protein interactions, details of protein-membrane interactions are poorly defined. Some of the best characterized examples are a phospholipase C pleckstrin homology domain specific for phosphatidylinositol trisphosphate (5) and small protein kinase-C-conserved (C2) domains specific for zwitterionic, particularly phosphatidylcholine membranes (6).Another group of proteins interacting with lipid membranes are pore-forming toxins (PFT) 1 (7-10), which bind to membranes before eliciting their toxic effects via the formation of transmembrane pores. The most studied PFT are bacterial since this group includes important virulence factors. Few examples of eukaryotic PFT have been well characterized, exceptions being the actinoporins, cytolysins found exclusively in sea anemones (10, 11). Members of this family have properties distinct from other PFT: they are composed of 175-179 amino acids, contain no cysteine residues, have pIϾ9.5, and show a preference for sphingomyelin (SM)-containing membranes. Actinoporins act on cellular and model lipid membranes by forming cation-selective pores with a hydrodynamic diameter of ϳ2 nm. The mechanism of pore formation involves at least two steps: binding of the water soluble m...