Properties, production, and applications of camelid single-domain antibody fragments

Camelids produce functional antibodies devoid of light chains of which the single N-terminal domain is fully capable of antigen binding. These single-domain antibody fragments (VHHs or Nanobodies®) have several advantages for biotechnological applications. They are well expressed in microorganisms and have a high stability and solubility. Furthermore, they are well suited for construction of larger molecules and selection systems such as phage, yeast, or ribosome display. This minireview offers an overview of (1) their properties as compared to conventional antibodies, (2) their production in microorganisms, with a focus on yeasts, and (3) their therapeutic applications

Alternative folding to a monomer or homopolymer is a common feature of the type 1 pilus subunit FimA from enteroinvasive bacteria

Adhesive type 1 pili from enteroinvasive, Gram-negative bacteria mediate attachment to host cells. Up to 3000 copies of the main pilus subunit, FimA, assemble into the filamentous, helical quaternary structure of the pilus rod via a mechanism termed donor-strand complementation, in which the N-terminal extension of each subunit, the donor strand, is inserted into the incomplete immunoglobulin-like fold of the preceding FimA subunit. For FimA from Escherichia coli, it has been previously shown that the protein can also adopt a monomeric, self-complemented conformation in which the donor strand is inserted intramolecularly in the opposite orientation relative to that observed for FimA polymers. Notably, soluble FimA monomers can act as apoptosis inhibitors in epithelial cells after uptake of type 1-piliated pathogens. Here, we show that the FimA orthologues from Escherichia coli, Shigella flexneri, and Salmonella enterica can all fold to form self-complemented monomers. We solved X-ray structures of all three FimA monomers at 0.89–1.69 Å resolutions, revealing identical, intramolecular donor-strand complementation mechanisms. Our results also showed that the pseudo-palindromic sequences of the donor strands in all FimA proteins permit their alternative folding possibilities. All FimA monomers proved to be 50–60 kJ/mol less stable against unfolding than their pilus rod-like counterparts (which exhibited very high energy barriers of unfolding and refolding). We conclude that the ability of FimA to adopt an alternative, monomeric state with anti-apoptotic activity is a general feature of FimA proteins of type 1-piliated bacteria.ISSN:0021-9258ISSN:1083-351

Complementation of DsbA deficiency with secreted thioredoxin variants reveals the crucial role of an efficient dithiol oxidant for catalyzed protein folding in the bacterial periplasm.

The thiol/disulfide oxidoreductase DsbA is the strongest oxidant of the thioredoxin superfamily and is required for efficient disulfide bond formation in the periplasm of Escherichia coli. To determine the importance of the redox potential of the final oxidant in periplasmic protein folding, we have investigated the ability of the most reducing thiol/disulfide oxidoreductase, E.coli thioredoxin, of complementing DsbA deficiency when secreted to the periplasm. In addition, we secreted thioredoxin variants with increased redox potentials as well as the catalytic a-domain of human protein disulfide isomerase (PDI) to the periplasm. While secreted wild-type thioredoxin and the most reducing thioredoxin variant could not replace DsbA, all more oxidizing thioredoxin variants as well as the PDI a-domain could complement DsbA deficiency in a DsbB-dependent manner. There is an excellent agreement between the activity of the secreted thioredoxin variants in vivo and their ability to oxidize polypeptides fast and quantitatively in vitro. We conclude that the redox potential of the direct oxidant of folding proteins and in particular its reactivity towards reduced polypeptides are crucial for efficient oxidative protein folding in the bacterial periplasm

Stochastic chain termination in bacterial pilus assembly

Adhesive type 1 pili from uropathogenic Escherichia coli strains are filamentous, supramolecular protein complexes consisting of a short tip fibrillum and a long, helical rod formed by up to several thousand copies of the major pilus subunit FimA. Here, we reconstituted the entire type 1 pilus rod assembly reaction in vitro, using all constituent protein subunits in the presence of the assembly platform FimD, and identified the so-far uncharacterized subunit FimI as an irreversible assembly terminator. We provide a complete, quantitative model of pilus rod assembly kinetics based on the measured rate constants of FimD-catalyzed subunit incorporation. The model reliably predicts the length distribution of assembled pilus rods as a function of the ratio between FimI and the main pilus subunit FimA and is fully consistent with the length distribution of membrane-anchored pili assembled in vivo. The results show that the natural length distribution of adhesive pili formed via the chaperone-usher pathway results from a stochastic chain termination reaction. In addition, we demonstrate that FimI contributes to anchoring the pilus to the outer membrane and report the crystal structures of (i) FimI in complex with the assembly chaperone FimC, (ii) the FimI-FimC complex bound to the N-terminal domain of FimD, and (iii) a ternary complex between FimI, FimA and FimC that provides structural insights on pilus assembly termination and pilus anchoring by FimI.ISSN:2041-172

The redox state regulates the conformation of Rv2466c to activate the antitubercular prodrug TP053

Rv2466c is a key oxidoreductase that mediates the reductive activation of TP053, a thienopyrimidine derivative that kills replicating and non-replicating Mycobacterium tuberculosis, but whose mode of action remains enigmatic. Rv2466c is a homodimer in which each subunit displays a modular architecture comprising a canonical thioredoxin fold with a Cys19-Pro20-Trp21-Cys22 motif, and an insertion consisting of a four \u3b1-helical bundle and a short \u3b1-helical hairpin. Strong evidence is provided for dramatic conformational changes during the Rv2466c redox cycle, which are essential for TP053 activity. Strikingly, a new crystal structure of the reduced form of Rv2466c revealed the binding of a C-terminal extension in \u3b1-helical conformation to a pocket next to the active site cysteine pair at the interface between the thioredoxin domain and the helical insertion domain. The ab initio low-resolution envelopes obtained from small angle X-ray scattering showed that the fully reduced form of Rv2466c adopts a 'closed' compact conformation in solution, similar to that observed in the crystal structure. In contrast, the oxidized form of Rv2466c displays an 'open' conformation, where tertiary structural changes in the \u3b1-helical subdomain suffice to account for the observed conformational transitions. Altogether our structural, biochemical and biophysical data strongly support a model in which the formation of the catalytic disulfide bond upon TP053 reduction triggers local structural changes that open the substrate binding site of Rv2466c allowing the release of the activated, reduced form of TP053. Our studies suggest that similar structural changes might have a functional role in other members of the thioredoxin-fold superfamily

Biomolecular solid-state NMR spectroscopy at 1200 MHz: the gain in resolution

Progress in NMR in general and in biomolecular applications in particular is driven by increasing magnetic-field strengths leading to improved resolution and sensitivity of the NMR spectra. Recently, persistent superconducting magnets at a magnetic field strength (magnetic induction) of 28.2 T corresponding to 1200 MHz proton resonance frequency became commercially available. We present here a collection of high-field NMR spectra of a variety of proteins, including molecular machines, membrane proteins, viral capsids, fibrils and large molecular assemblies. We show this large panel in order to provide an overview over a range of representative systems under study, rather than a single best performing model system. We discuss both carbon-13 and proton-detected experiments, and show that in C-13 spectra substantially higher numbers of peaks can be resolved compared to 850 MHz while for H-1 spectra the most impressive increase in resolution is observed for aliphatic side-chain resonances.ISSN:0925-2738ISSN:1573-500