Limited tolerance towards folded elements during secretion of the autotransporter Hbp

Many virulence factors secreted by pathogenic Gram-negative bacteria belong to the autotransporter (AT) family. ATs consist of a passenger domain, which is the actual secreted moiety, and a β-domain that facilitates the transfer of the passenger domain across the outer membrane. Here, we analysed folding and translocation of the AT passenger, using Escherichia coli haemoglobin protease (Hbp) as a model protein. Dual cysteine mutagenesis, instigated by the unique crystal structure of the Hbp passenger, resulted in intramolecular disulphide bond formation dependent on the periplasmic enzyme DsbA. A small loop tied off by a disulphide bond did not interfere with secretion of Hbp. In contrast, a bond between different domains of the Hbp passenger completely blocked secretion resulting in degradation by the periplasmic protease DegP. In the absence of DegP, a translocation intermediate accumulated in the outer membrane. A similar jammed intermediate was formed upon insertion of a calmodulin folding moiety into Hbp. The data suggest that Hbp can fold in the periplasm but must retain a certain degree of flexibility and/or modest width to allow translocation across the outer membrane

Design of tryptophan-containing mutants of the symmetrical Pizza protein for biophysical studies

International audienceβ-propeller proteins are highly symmetrical, being composed of a repeated motif with four anti-parallel β-sheets arranged around a central axis. Recently we designed the first completely symmetrical β-propeller protein, Pizza6, consisting of six identical tandem repeats. Pizza6 is expected to prove a useful building block for bionanotechnology, and also a tool to investigate the folding and evolution of β-propeller proteins. Folding studies are made difficult by the high stability and the lack of buried Trp residues to act as monitor fluorophores, so we have designed and characterized several Trp-containing Pizza6 derivatives. In total four proteins were designed, of which three could be purified and characterized. Crystal structures confirm these mutant proteins maintain the expected structure, and a clear redshift of Trp fluorescence emission could be observed upon denaturation. Among the derivative proteins, Pizza6-AYW appears to be the most suitable model protein for future folding/unfolding kinetics studies as it has a comparable stability as natural β-propeller proteins

Crystal structures of penicillin-binding protein 3 (PBP3) from methicillin-resistant staphylococcus aureus in the apo and cefotaxime‐bound forms

Staphylococcus aureus is a widespread Gram‐positive opportunistic pathogen, and a methicillin‐resistant form (MRSA) is particularly difficult to treat clinically. We have solved two crystal structures of penicillin‐binding protein (PBP) 3 (PBP3) from MRSA, the apo form and a complex with the β-lactam antibiotic cefotaxime, and used electrospray mass spectrometry to measure its sensitivity to a variety of penicillin derivatives. PBP3 is a class B PBP, possessing an N-terminal non-penicillin‐binding domain, sometimes called a dimerization domain, and a C-terminal transpeptidase domain. The model shows a different orientation of its two domains compared to earlier models of other class B PBPs and a novel, larger N-domain. Consistent with the nomenclature of “dimerization domain”, the N-terminal region forms an apparently tight interaction with a neighboring molecule related by a 2-fold symmetry axis in the crystal structure. This dimer form is predicted to be highly stable in solution by the PISA server, but mass spectrometry and analytical ultracentrifugation provide unequivocal evidence that the protein is a monomer in solution

Evolution and molecular basis of a novel allosteric property of crocodilian hemoglobin

The extraordinary breath-hold diving capacity of crocodilians has been ascribed to a unique mode of allosterically regulating hemoglobin (Hb)-oxygenation in circulating red blood cells. We investigated the origin and mechanistic basis of this novel biochemical phenomenon by performing directed mutagenesis experiments on resurrected ancestral Hbs. Comparisons of Hb function between the common ancestor of archosaurs (the group that includes crocodilians and birds) and the last common ancestor of modern crocodilians revealed that regulation of Hb-O2 affinity via allosteric binding of bicarbonate ions represents a croc-specific innovation that evolved in combination with the loss of allosteric regulation by ATP binding. Mutagenesis experiments revealed that evolution of the novel allosteric function in crocodilians and the concomitant loss of ancestral function were not mechanistically coupled and were caused by different sets of substitutions. The gain of bicarbonate sensitivity in crocodilian Hb involved the direct effect of few amino acid substitutions at key sites in combination with indirect effects of numerous other substitutions at structurally disparate sites. Such indirect interaction effects suggest that evolution of the novel protein function was conditional on neutral mutations that produced no adaptive benefit when they first arose but that contributed to a permissive background for subsequent function-altering mutations at other sites. Due to the context dependence of causative substitutions, the unique allosteric properties of crocodilian Hb cannot be easily transplanted into divergent homologs of other species

Crystal structure of a novel polyisoprenoid-binding protein from Thermus thermophilus HB8

The isoprenoid quinones exist widely among prokaryotes and eukaryotes. They play essential roles in respiratory electron transport and in controlling oxidative stress and gene regulation. In the isoprenoid quinone biosynthetic pathway, polyprenyl pyrophosphates are used as isoprenoid side-chain precursors. Here we report the crystal structure of a novel polyprenyl pyrophosphate binding protein, TT1927b, from Thermus thermophilus HB8, complexed with its ligand. This protein belongs to the YceI-like family in the Pfam database, and its sequence homologs are present in a broad range of bacteria and archaea. The structure consists of an extended, eight-stranded, antiparallel β-barrel. In the hydrophobic pore of the barrel, the protein binds the polyisoprenoid chain by hydrophobic interactions. Its overall structure resembles the lipocalin fold, but there is no sequence homology between TT1927b and the lipocalin family of proteins

Intersubunit linker length as a modifier of protein stability: Crystal structures and thermostability of mutant TRAP

The ability of proteins to self-assemble into complex, functional nanoscale structures is expected to become of significant use in the manufacture of artificial nanodevices with a wide range of novel applications. The bacterial protein TRAP has potential uses as a nanoscale component as it is ring-shaped, with a central, modifiable cavity. Furthermore, it can be engineered to make a ring of 12-fold symmetry, which is advantageous for packing into two-dimensional arrays. The 12mer form of TRAP is made by linking multiple subunits together on the same polypeptide, but the usefulness of the 12mers described to date is limited by their poor stability. Here we show that, by altering the length of the peptide linker between subunits, the thermostability can be significantly improved. Since the subunit interfaces of the different 12mers are essentially identical, stabilization arises from the reduction of strain in the linkers. Such a simple method of controlling the stability of modular proteins may have wide applications, and demonstrates the lack of absolute correlation between interactions observable by crystallography and the internal energy of a complex