Corrigendum: A New Strain Collection for Improved Expression of Outer Membrane Proteins.

[This corrects the article DOI: 10.3389/fcimb.2017.00464.].

Insights into the autotransport process of a trimeric autotransporter, Yersinia Adhesin A (YadA).

Trimeric autotransporter adhesins (TAAs) are a subset of a larger protein family called the type V secretion systems. They are localized on the cell surface of Gram-negative bacteria, function as mediators of attachment to inorganic surfaces and host cells, and thus include important virulence factors. Yersinia adhesin A (YadA) from Yersinia enterocolitica is a prototypical TAA that is used extensively to study the structure and function of the type Vc secretion system. A solid-state NMR study of the membrane anchor domain of YadA previously revealed a flexible stretch of small residues, termed the ASSA region, that links the membrane anchor to the stalk domain. In this study, we present evidence that single amino acid proline substitutions produce two different conformers of the membrane anchor domain of YadA; one with the N-termini facing the extracellular surface, and a second with the N-termini located in the periplasm. We propose that TAAs adopt a hairpin intermediate during secretion, as has been shown before for other subtypes of the type V secretion system. As the YadA transition state intermediate can be isolated from the outer membrane, future structural studies should be possible to further unravel details of the autotransport process.

A New Strain Collection for Improved Expression of Outer Membrane Proteins.

Almost all integral membrane proteins found in the outer membranes of Gram-negative bacteria belong to the transmembrane ß-barrel family. These proteins are not only important for nutrient uptake and homeostasis, but are also involved in such processes as adhesion, protein secretion, biofilm formation, and virulence. As surface exposed molecules, outer membrane ß-barrel proteins are also potential drug and vaccine targets. High production levels of heterologously expressed proteins are desirable for biochemical and especially structural studies, but over-expression and subsequent purification of membrane proteins, including outer membrane proteins, can be challenging. Here, we present a set of deletion mutants derived from E. coli BL21 Gold (DE3) designed for the over-expression of recombinant outer membrane proteins. These strains harbor deletions of four genes encoding abundant ß-barrel proteins in the outer membrane (OmpA, OmpC, OmpF, and LamB), both single and in all combinations of double, triple, and quadruple knock-outs. The sequences encoding these outer membrane proteins were deleted completely, leaving only a minimal scar sequence, thus preventing the possibility of genetic reversion. Expression tests in the quadruple mutant strain with four test proteins, including a small outer membrane ß-barrel protein and variants thereof as well as two virulence-related autotransporters, showed significantly improved expression and better quality of the produced proteins over the parent strain. Differences in growth behavior and aggregation in the presence of high salt were observed, but these phenomena did not negatively influence the expression in the quadruple mutant strain when handled as we recommend. The strains produced in this study can be used for outer membrane protein production and purification, but are also uniquely useful for labeling experiments for biophysical measurements in the native membrane environment.

Yersinia adhesins: An arsenal for infection.

The Yersiniae are a group of Gram-negative coccobacilli inhabiting a wide range of habitats. The genus harbors three recognized human pathogens: Y. enterocolitica and Y. pseudotuberculosis, which both cause gastrointestinal disease, and Y. pestis, the causative agent of plague. These three organisms have served as models for a number of aspects of infection biology, including adhesion, immune evasion, evolution of pathogenic traits, and retracing the course of ancient pandemics. The virulence of the pathogenic Yersiniae is heavily dependent on a number of adhesin molecules. Some of these, such as the Yersinia adhesin A and invasin of the enteropathogenic species, and the pH 6 antigen of Y. pestis, have been extensively studied. However, genomic sequencing has uncovered a host of other adhesins present in these organisms, the functions of which are only starting to be investigated. Here, we review the current state of knowledge on the adhesin molecules present in the Yersiniae, and their functions and putative roles in the infection process.

Type V Secretion Systems in Bacteria.

Type V secretion denotes a variety of secretion systems that cross the outer membrane in Gram-negative bacteria but that depend on the Sec machinery for transport through the inner membrane. They are possibly the simplest bacterial secretion systems, because they consist only of a single polypeptide chain (or two chains in the case of two-partner secretion). Their seemingly autonomous transport through the outer membrane has led to the term "autotransporters" for various subclasses of type V secretion. In this chapter, we review the structure and function of these transporters and review recent findings on additional factors involved in the secretion process, which have put the term "autotransporter" to debate.

Solid-state NMR Study of the YadA Membrane-Anchor Domain in the Bacterial Outer Membrane.

MAS-NMR was used to study the structure and dynamics at ambient temperatures of the membrane-anchor domain of YadA (YadA-M) in a pellet of the outer membrane of E. coli in which it was expressed. YadA is an adhesin from the pathogen Yersinia enterocolitica that is involved in interactions with the host cell, and it is a model protein for studying the autotransport process. Existing assignments were sucessfully transferred to a large part of the YadA-M protein in the E. coli lipid environment by using (13) C-(13) C DARR and PDSD spectra at different mixing times. The chemical shifts in most regions of YadA-M are unchanged relative to those in microcrystalline YadA-M preparations from which a structure has previously been solved, including the ASSA region that is proposed to be involved in transition-state hairpin formation for transport of the soluble domain. Comparisons of the dynamics between the microcrystalline and membrane-embedded samples indicate greater flexibility of the ASSA region in the outer-membrane preparation at physiological temperatures. This study will pave the way towards MAS-NMR structure determination of membrane proteins, and a better understanding of functionally important dynamic residues in native membrane environments.

Analysis of dimerization of BTB-IVR domains of Keap1 and its interaction with Cul3, by molecular modeling.

Oxidative damage has been associated with various neurodegenerative diseases including Parkinson's disease, amyotrophic lateral sclerosis (ALS), and Alzheimer's disease, as well as non-neurodegenerative conditions such as cancer and heart disease. The Keap1-Nrf2 system plays a central role in the protection of cells against oxidative and xenobiotic stress. The Nrf2 transcription function and its degradation by the proteasomal pathway (Keap1-Nrf2-Cul3-Roc1 complex) are regulated by the cytoplasmic repressor protein, Keap1 which possesses BTB, BACK (IVR region) and Kelch domains. The BTB-BACK domains are important for Keap1 homo-dimerization as well as to interact with Cullin-3 for Nrf2 degradation. The crystal structure of the Keap1-Kelch domain is known; however, that of the BTB-BACK domains are not yet determined. We present here, through molecular modeling studies, the analysis of Keap1-BTB dimerization, and of BTB-BACK domains role in complex with Cul3. The electrostatic charge distribution at the BTB dimer interface of Keap1 is significantly different from other known BTB containing protein structures. Another intriguing feature is also observed that the non-conserved residues at the BTB-BACK-Cul3 interface region may play critical role for differentiating Cul3 recognition by Keap1 from other adaptor proteins for their specific substrates proteasomal degradation.