A conserved β-bulge glycine residue facilitates folding and increases stability of the mouse α-defensin cryptdin-4

Defensins are key components of both innate and adaptive immune responses to pathogens. Cryptdins are mouse alpha-defensins that are secreted from Paneth cells in the small intestine and have disulfide-stabilised structures and antibacterial activities against both Gram-positive and Gram-negative bacteria. The folding and three-dimensional structures of alpha-defensins are thought to depend on a conserved glycine residue that forms a β-bulge. Here we investigated the role of this conserved glycine at position 19 of cryptdin-4 (Crp4) in terms of the folding, structure and stability. A Crp4 variant with D-Ala at position 19 folded efficiently, was stabilised by a large number of hydrogen bonds, and resisted proteolysis in simulated intestinal fluid. Although a variant with L-Ala at position 19 was able to adopt the correct fold, it showed less efficient folding and was degraded more rapidly than the D-Ala variant. These results demonstrate the key role that glycine residues can have in folding of bioactive peptides and can provide insights to guide design of stable antimicrobial peptides that fold efficiently.

The archaeal protein SepF is essential for cell division in Haloferax volcanii

In most bacteria, cell division depends on the tubulin homolog FtsZ and other proteins, such as SepF, that form a complex termed the divisome. Cell division also depends on FtsZ in many archaea, but other components of the divisome are unknown. Here, we demonstrate that a SepF homolog plays important roles in cell division in Haloferax volcanii, a halophilic archaeon that is known to have two FtsZ homologs with slightly different functions (FtsZ1 and FtsZ2). SepF co-localizes with both FtsZ1 and FtsZ2 at midcell. Attempts to generate a sepF deletion mutant were unsuccessful, suggesting an essential role. Indeed, SepF depletion leads to severe cell division defects and formation of large cells. Overexpression of FtsZ1-GFP or FtsZ2-GFP in SepF-depleted cells results in formation of filamentous cells with a high number of FtsZ1 rings, while the number of FtsZ2 rings is not affected. Pull-down assays support that SepF interacts with FtsZ2 but not with FtsZ1, although SepF appears delocalized in the absence of FtsZ1. Archaeal SepF homologs lack a glycine residue known to be important for polymerization and function in bacteria, and purified H. volcanii SepF forms dimers, suggesting that polymerization might not be important for the function of archaeal SepF.

Archaeal SepF is essential for cell division in Haloferax volcanii

Bacterial cell division has been studied for decades but reports on the different archaeal cell division systems are rare. In many archaea, cell division depends on the tubulin homolog FtsZ, but further components of the divisome in these archaea are unknown. The halophilic archaeon Haloferax volcanii encodes two FtsZ homologs with different functions in cell division and a putative SepF homolog. In bacteria, SepF is part of the divisome and is recruited early to the FtsZ ring, where it most likely stimulates FtsZ ring formation. H. volcanii SepF co-localized with FtsZ1 and FtsZ2 at midcell. Overexpression of SepF had no effect on cell morphology, but no sepF deletion mutants could be generated. SepF depletion led to a severe cell division defect, resulting in cells with a strongly increased size. Overexpression of FtsZ1- and FtsZ2-GFP in SepF-depleted cells resulted in filamentous cells with an increasing number of FtsZ1 rings depending on the cell length, whereas FtsZ2 rings were not increased. Pull-down assays with HA-tagged SepF identified an interaction with FtsZ2 but not with FtsZ1. Archaeal SepF homologs lack the conserved glycine residue important for polymerization in bacteria and the H. volcanii SepF was purified as a dimer, suggesting that in contrast to the bacterial SepF homologs, polymerization does not seem to be important for its function. A model is proposed where first the FtsZ1 ring is formed and where SepF recruits FtsZ2 to the FtsZ1 ring, resulting in the formation of the FtsZ2 ring. This study provides important novel insights into cell division in archaea and shows that SepF is an important part of the divisome in FtsZ containing archaea.

Glycine in Water Favors the Polyproline II State

Conformational preferences of amino acid residues in water are determined by the backbone and side-chain properties. Alanine is known for its high polyproline II (pPII) propensity. The question of relative contributions of the backbone and side chain to the conformational preferences of alanine and other amino acid residues in water is not fully resolved. Because glycine lacks a heavy-atom side chain, glycine-based peptides can be used to examine to which extent the backbone properties affect the conformational space. Here, we use published spectroscopic data for the central glycine residue of cationic triglycine in water to demonstrate that its conformational space is dominated by the pPII state. We assess three commonly used molecular dynamics (MD) force fields with respect to their ability to capture the conformational preferences of the central glycine residue in triglycine. We show that pPII is the mesostate that enables the functional backbone groups of the central residue to form the most hydrogen bonds with water. Our results indicate that the pPII propensity of the central glycine in GGG is comparable to that of alanine in GAG, implying that the water-backbone hydrogen bonding is responsible for the high pPII content of these residues.

Unexpected Enhancement of HDACs Inhibition by MeS Substitution at C-2 Position of Fluoro Largazole

Given our previous finding that fluorination at the C18 position of largazole showed reasonably good tolerance towards inhibitory activity and selectivity of histone deacetylases (HDACs), further modification on the valine residue in the fluoro-largazole’s macrocyclic moiety with S-Me l-Cysteine or Glycine residue was performed. While the Glycine-modified fluoro analog showed poor activity, the S-Me l-Cysteine-modified analog emerged to be a very potent HDAC inhibitor. Unlike all previously reported C2-modified compounds in the largazole family (including our recent fluoro-largazole analogs) where replacement of the Val residue has failed to provide any potency improvement, the S-Me l-Cysteine-modified analog displayed significantly enhanced (five–nine-fold) inhibition of all the tested HDACs while maintaining the selectivity of HDAC1 over HDAC6, as compared to largazole thiol. A molecular modeling study provided rational explanation and structural evidence for the enhanced inhibitory activity. This new finding will aid the design of novel potent HDAC inhibitors.

A native prokaryotic voltage-dependent calcium channel with a novel selectivity filter sequence

Voltage-dependent Ca2+ channels (Cavs) are indispensable for coupling action potentials with Ca2+ signaling in living organisms. The structure of Cavs is similar to that of voltage-dependent Na+ channels (Navs). It is known that prokaryotic Navs can obtain Ca2+ selectivity by negative charge mutations of the selectivity filter, but native prokaryotic Cavs had not yet been identified. We report the first identification of a native prokaryotic Cav, CavMr, whose selectivity filter contains a smaller number of negatively charged residues than that of artificial prokaryotic Cavs. A relative mutant whose selectivity filter was replaced with that of CavMr exhibits high Ca2+ selectivity. Mutational analyses revealed that the glycine residue of the CavMr selectivity filter is a determinant for Ca2+ selectivity. This glycine residue is well conserved among subdomains I and III of eukaryotic Cavs. These findings provide new insight into the Ca2+ selectivity mechanism that is conserved from prokaryotes to eukaryotes.