The cavity-chaperone Skp protects its substrate from aggregation but allows independent folding of substrate domains.

Outer membrane proteins (OMPs) of gram-negative bacteria are synthesized in the cytosol and must cross the periplasm before insertion into the outer membrane. The 17-kDa protein (Skp) is a periplasmic chaperone that assists the folding and insertion of many OMPs, including OmpA, a model OMP with a membrane embedded beta-barrel domain and a periplasmic alphabeta domain. Structurally, Skp belongs to a family of cavity-containing chaperones that bind their substrates in the cavity, protecting them from aggregation. However, some substrates, such as OmpA, exceed the capacity of the chaperone cavity, posing a mechanistic challenge. Here, we provide direct NMR evidence that, while bound to Skp, the beta-barrel domain of OmpA is maintained in an unfolded state, whereas the periplasmic domain is folded in its native conformation. Complementary cross-linking and NMR relaxation experiments show that the OmpA beta-barrel is bound deep within the Skp cavity, whereas the folded periplasmic domain protrudes outside of the cavity where it tumbles independently from the rest of the complex. This domain-based chaperoning mechanism allows the transport of beta-barrels across the periplasm in an unfolded state, which may be important for efficient insertion into the outer membrane.

Anti-obesity and anti-tumor pro-apoptotic peptides are sufficient to cause release of cytochrome c from vesicles.

Peptides that target tissue for an apoptotic death have potential as therapeutics in a variety of disease conditions. The class of peptides described herein enters the cell through a specific receptor-mediated interaction. Once inside the cell, the peptide migrates toward the mitochondria, where the membrane barrier is disrupted. These experiments demonstrate that upon treatment with these short peptides large unilamellar vesicles are not lysed, a graded mode of leakage is observed and the transient pores formed by these peptides are large enough to release entrapped cytochrome c from the vesicles.

EC144 is a potent inhibitor of the heat shock protein 90.

Alkyne 40, 5-(2-amino-4-chloro-7-((4-methoxy-3,5-dimethylpyridin-2-yl)methyl)-7H-pyrrolo[2,3-d]pyrimidin-5-yl)-2-methylpent-4-yn-2-ol (EC144), is a second generation inhibitor of heat shock protein 90 (Hsp90) and is substantially more potent in vitro and in vivo than the first generation inhibitor 14 (BIIB021) that completed phase II clinical trials. Alkyne 40 is more potent than 14 in an Hsp90α binding assay (IC(50) = 1.1 vs 5.1 nM) as well as in its ability to degrade Her-2 in MCF-7 cells (EC(50) = 14 vs 38 nM). In a mouse model of gastric tumors (N87), 40 stops tumor growth at 5 mg/kg and causes partial tumor regressions at 10 mg/kg (po, qd × 5). Under the same conditions, 14 stops tumor growth only at 120 mg/kg, and does not induce partial regressions. Thus, alkyne 40 is approximately 20-fold more efficacious than 14 in mice.

Crystal structure of BamD: an essential component of the ß-Barrel assembly machinery of gram-negative bacteria.

Folding and insertion of integral ß-barrel proteins in the outer membrane (OM) is an essential process for Gram-negative bacteria that requires the ß-barrel assembly machinery (BAM). Efficient OM protein (OMP) folding and insertion appears to require a consensus C-terminal signal in OMPs characterized by terminal F or W residues. The BAM complex is embedded in the OM and, in Escherichia coli, consists of the ß-barrel BamA and four lipoproteins BamBCDE. BamA and BamD are broadly distributed across all species of Gram-negative bacteria, whereas the other components are present in only a subset of species. BamA and BamD are also essential for viability, suggesting that these two proteins constitute the functional core of the bacterial BAM complex. Here, we present the crystal structure of BamD from the thermophilic bacteria Rhodothermus marinus refined to 2.15 Å resolution. The protein contains five tetratricopeptide repeats (TPRs) organized into two offset tandems, each capped by a terminal helix. The N-terminal domain contains three TPRs and displays remarkable structural similarity with proteins that recognize targeting signals in extended conformations. The C-terminal domain harbors the remaining two TPRs and previously described mutations that impair binding to other BAM components map to this domain. Therefore, the structure suggests a model where the C-terminal domain provides a scaffold for interaction with BAM components, while the N-terminal domain participates in interaction with the substrates, either recognizing the C-terminal consensus sequence or binding unfolded OMP intermediates.

Structural evaluation of a novel pro-apoptotic peptide coupled to CNGRC tumor homing sequence by NMR.

Hunter-killer peptides (HKPs) are synthetic peptides that target specific cell types for apoptosis. These studies report functional and structural characteristics of HKP9, an hunter-killer peptide that specifically targets tumor vasculature with a new apoptotic sequence. Vesicle leakage experiments were performed as a model for membrane perturbing activity. Placement of the homing sequence reduces both cell toxicity and vesicle leakage activity. NMR studies elucidate the conformation and orientation of HKP9 in micelles. The positively charged end of the HKP9 killing sequence is solvent exposed; however, the central portion of the peptide is helical and buried in dodecylphosphorylcholine micelles. The homing sequence is less solvent exposed than in a previously reported tumor-homing peptide. The results suggest that solvent accessibility of the homing sequence should be considered in design of future peptides.