Inhibitors for membrane transporters have been shown to be indispensable as drugs and tool compounds. The proton-dependent oligopeptide transporters PEPT1 and PEPT2 from the SLC15 family play important roles in human and mammalian physiology. With Lys[Z(NO₂)]-Val (LZNV), a modified Lys-Val dipeptide, a potent transport inhibitor for PEPT1 and PEPT2 is available. Here we present the crystal structure of the peptide transporter YePEPT in complex with LZNV. The structure revealed the molecular interactions for inhibitor binding and a previously undescribed mostly hydrophobic pocket, the PZ pocket, involved in interaction with LZNV. Comparison with a here determined ligand-free structure of the transporter unveiled that the initially absent PZ pocket emerges through conformational changes upon inhibitor binding. The provided biochemical and structural information constitutes an important framework for the mechanistic understanding of inhibitor binding and action in proton-dependent oligopeptide transporters.

Human heteromeric amino acid transporters (HATs) are membrane protein complexes that facilitate the transport of specific amino acids across cell membranes. Loss of function or overexpression of these transporters is implicated in several human diseases such as renal aminoacidurias and cancer. HATs are composed of two subunits, a heavy and a light subunit, that are covalently connected by a disulphide bridge. Light subunits catalyse amino acid transport and consist of twelve transmembrane α-helix domains. Heavy subunits are type II membrane N-glycoproteins with a large extracellular domain and are involved in the trafficking of the complex to the plasma membrane. Structural information on HATs is scarce because of the difficulty in heterologous overexpression. Recently, we had a major breakthrough with the overexpression of a recombinant HAT, 4F2hc-LAT2, in the methylotrophic yeast Pichia pastoris. Microgram amounts of purified protein made possible the reconstruction of the first 3D map of a human HAT by negative-stain transmission electron microscopy. Here we report the important stabilization of purified human 4F2hc-LAT2 using a combination of two detergents, i.e., n-dodecyl-β-D-maltopyranoside and lauryl maltose neopentyl glycol, and cholesteryl hemisuccinate. The superior quality and stability of purified 4F2hc-LAT2 allowed the measurement of substrate binding by scintillation proximity assay. In addition, an improved 3D map of this HAT could be obtained. The detergent-induced stabilization of the purified human 4F2hc-LAT2 complex presented here paves the way towards its crystallization and structure determination at high-resolution, and thus the elucidation of the working mechanism of this important protein complex at the molecular level.

The light-driven proton pump bacteriorhodopsin (BR) from the extreme halophilic archaeon Halobacterium salinarum is a retinal-binding protein, which forms highly ordered and thermally stable 2D crystals in native membranes (termed purple membranes). BR and purple membranes (PMs) have been and are still being intensively studied by numerous researchers from different scientific disciplines. Furthermore, PMs are being successfully used in new, emerging technologies such as bioelectronics and bionanotechnology. Most published studies used the wild-type form of BR, because of the intrinsic difficulty to produce genetically modified versions in purple membranes homologously. However, modification and engineering is crucial for studies in basic research and, in particular, to tailor BR for specific applications in applied sciences. We present an extensive and detailed protocol ranging from the genetic modification and cultivation of H. salinarum to the isolation, and biochemical, biophysical and functional characterization of BR and purple membranes. Pitfalls and problems of the homologous expression of BR versions in H. salinarum are discussed and possible solutions presented. The protocol is intended to facilitate the access to genetically modified BR versions for researchers of different scientific disciplines, thus increasing the application of this versatile biomaterial.

Synthetic biology approaches range from the introduction of unique features into organisms to the assembly of isolated biomacromolecules or synthetic building blocks into artificial biological systems with biomimetic or completely novel functionalities. Simple molecular systems can be based on containers on the nanoscale that are equipped with tailored functional modules for various applications in healthcare, industry or biological and medical research. The concept, or vision, of assembling native or engineered proteins and/or synthetic components as functional modules into molecular systems is discussed. The main focus is laid on the engineering of energizing modules generating chemical energy, transport modules using this energy to translocate molecules between compartments of a molecular system, and catalytic modules (bio-)chemically processing the molecules. Further key aspects of this discourse are possible approaches for the assembly of simple nanofactories and their applications in biotechnology and medical health.

Datasets to Stauffer et al. (2021) Sci. Rep.