Transmembrane domains interactions within the membrane milieu: Principles, advances and challenges

AbstractProtein–protein interactions within the membrane are involved in many vital cellular processes. Consequently, deficient oligomerization is associated with known diseases. The interactions can be partially or fully mediated by transmembrane domains (TMD). However, in contrast to soluble regions, our knowledge of the factors that control oligomerization and recognition between the membrane–embedded domains is very limited. Due to the unique chemical and physical properties of the membrane environment, rules that apply to interactions between soluble segments are not necessarily valid within the membrane. This review summarizes our knowledge on the sequences mediating TMD–TMD interactions which include conserved motifs such as the GxxxG, QxxS, glycine and leucine zippers, and others. The review discusses the specific role of polar, charged and aromatic amino acids in the interface of the interacting TMD helices. Strategies to determine the strength, dynamics and specificities of these interactions by experimental (ToxR, TOXCAT, GALLEX and FRET) or various computational approaches (molecular dynamic simulation and bioinformatics) are summarized. Importantly, the contribution of the membrane environment to the TMD–TMD interaction is also presented. Studies utilizing exogenously added TMD peptides have been shown to influence in vivo the dimerization of intact membrane proteins involved in various diseases. The chirality independent TMD–TMD interactions allows for the design of novel short d- and l-amino acids containing TMD peptides with advanced properties. Overall these studies shed light on the role of specific amino acids in mediating the assembly of the TMDs within the membrane environment and their contribution to protein function. This article is part of a Special Issue entitled: Protein Folding in Membranes

Deciphering the Mechanical Properties of Type III Secretion System EspA Protein by Single Molecule Force Spectroscopy

Bacterial pathogens inject virulence factors into host cells during bacterial infections using type III secretion systems. In enteropathogenic <i>Escherichia coli</i>, this system contains an external filament, formed by a self-oligomerizing protein called <i>E. coli</i> secreted protein A (EspA). The EspA filament penetrates the thick viscous mucus layer to facilitate the attachment of the bacteria to the gut-epithelium. To do that, the EspA filament requires noteworthy mechanical endurance considering the mechanical shear stresses found within the intestinal tract. To date, the mechanical properties of the EspA filament and the structural and biophysical knowledge of monomeric EspA are very limited, mostly due to the strong tendency of the protein to self-oligomerize. To overcome this limitation, we employed a single molecule force spectroscopy (SMFS) technique and studied the mechanical properties of EspA. Force extension dynamic of (I91)₄-EspA-(I91)₄ chimera revealed two structural unfolding events occurring at low forces during EspA unfolding, thus indicating no unique mechanical stability of the monomeric protein. SMFS examination of purified monomeric EspA protein, treated by a gradually refolding protocol, exhibited similar mechanical properties as the EspA protein within the (I91)₄-EspA-(I91)₄ chimera. Overall, our results suggest that the mechanical integrity of the EspA filament likely originates from the interactions between EspA monomers and not from the strength of an individual monomer

A highly effective component vaccine against nontyphoidal salmonella enterica infections

Nontyphoidal Salmonella enterica (NTS) infections are a major burden to global public health, as they lead to diseases ranging from gastroenteritis to systemic infections and there is currently no vaccine available. Here, we describe a highly effective component vaccine against S. enterica serovar Typhimurium in both gastroenteritis and systemic murine infection models. We devised an approach to generate supernatants of S. enterica serovar Typhimurium, an organism that is highly abundant in virulence factors. Immunization of mice with this supernatant resulted in dramatic protection against a challenge with serovar Typhimurium, showing increased survival in the systemic model and decreased intestinal pathology in the gastrointestinal model. Protection correlated with specific IgA and IgG levels in the serum and specific secretory IgA levels in the feces of immunized mice. Initial characterization of the protective antigens in the bacterial culture supernatants revealed a subset of antigens that exhibited remarkable stability, a highly desirable characteristic of an effective vaccine to be used under suboptimal environmental conditions in developing countries. We were able to purify a subset of the peptides present in the supernatants and show their potential for immunization of mice against serovar Typhimurium resulting in a decreased level of colonization. This component vaccine shows promise with regard to protecting against NTS, and further work should significantly help to establish vaccines against these prevalent infections.Peer reviewed: YesNRC publication: Ye