Interdomain communication in a homodimeric ABC transporter.

ABC transporters are found in all organisms and almost every cellular compartment. They mediate the transport of various solutes across membranes, energized by ATP binding and hydrolysis. Dysfunctions can result in severe diseases, such as cystic fibrosis or antibiotic resistance.

Insight into the Structural Basis for Dual Nucleic Acid-Recognition by the Scaffold Attachment Factor B2 Protein.

The family of scaffold attachment factor B (SAFB) proteins comprises three members and was first identified as binders of the nuclear matrix/scaffold. Over the past two decades, SAFBs were shown to act in DNA repair, mRNA/(l)ncRNA processing and as part of protein complexes with chromatin-modifying enzymes. SAFB proteins are approximately 100 kDa-sized dual nucleic acid-binding proteins with dedicated domains in an otherwise largely unstructured context, but whether and how they discriminate DNA and RNA binding has remained enigmatic.

Unidirectional mannitol synthesis of MtlD is facilitated by the helix-loop-helix-mediated dimer formation.

Persistence of Acinetobacter baumannii in environments with low water activity is largely attributed to the biosynthesis of compatible solutes. Mannitol is one of the key compatible solutes in A. baumannii, and it is synthesized by a bifunctional mannitol-1-phosphate dehydrogenase/phosphatase (AbMtlD).

Large-Scale Recombinant Production of the SARS-CoV-2 Proteome for High-Throughput and Structural Biology Applications.

The highly infectious disease COVID-19 caused by the SARS-CoV-2 poses a severe threat to humanity and demands the redirection of scientific efforts and criteria to organized research projects. The international consortium seeks to provide such new approaches by gathering scientific expertise worldwide. In particular, making available viral proteins and RNAs will pave the way to understanding the SARS-CoV-2 molecular components in detail.

Spatial Location in Integrated Circuits through Infrared Microscopy.

In this paper, we present an infrared microscopy based approach for structures' location in integrated circuits, to automate their secure characterization. The use of an infrared sensor is the key device for internal integrated circuit inspection. Two main issues are addressed.

H, C, and N backbone chemical shift assignments of coronavirus-2 non-structural protein Nsp10.

The international Covid19-NMR consortium aims at the comprehensive spectroscopic characterization of SARS-CoV-2 RNA elements and proteins and will provide NMR chemical shift assignments of the molecular components of this virus. The SARS-CoV-2 genome encodes approximately 30 different proteins. Four of these proteins are involved in forming the viral envelope or in the packaging of the RNA genome and are therefore called structural proteins.

An inventory of lysosomal ABC transporters.

ABC transporters fulfill diverse physiological functions in different cellular localizations ranging from the plasma membrane to intracellular membranous compartments. Several ABC transporters have been spotted in the endolysosomal system, which consists of endosomes, autophagosomes, lysosomes, and lysosome-related organelles. In this review, we present an overview of lysosomal ABC transporters including ABCA2, ABCA3, ABCA5, ABCB6, ABCB9, and ABCD4, discussing their trafficking routes, putative substrates, potential physiological functions, and associated diseases.

Peptide translocation by the lysosomal ABC transporter TAPL is regulated by coupling efficiency and activation energy.

The lysosomal polypeptide transporter TAPL belongs to the superfamily of ATP-binding cassette transporters. TAPL forms a homodimeric transport complex, which translocates oligo- and polypeptides into the lumen of lysosomes driven by ATP hydrolysis. Although the structure and the function of ABC transporters were intensively studied in the past, details about the single steps of the transport cycle are still elusive.

Lysosomal targeting of the ABC transporter TAPL is determined by membrane-localized charged residues.

The human lysosomal polypeptide ABC transporter TAPL (ABC subfamily B member 9, ABCB9) transports 6-59-amino-acid-long polypeptides from the cytosol into lysosomes. The subcellular localization of TAPL depends solely on its N-terminal transmembrane domain, TMD0, which lacks conventional targeting sequences. However, the intracellular route and the molecular mechanisms that control TAPL localization remain unclear.

Structural and functional insights into the interaction and targeting hub TMD0 of the polypeptide transporter TAPL.

The ATP-binding cassette transporter TAPL translocates polypeptides from the cytosol into the lysosomal lumen. TAPL can be divided into two functional units: coreTAPL, active in ATP-dependent peptide translocation, and the N-terminal membrane spanning domain, TMD0, responsible for cellular localization and interaction with the lysosomal associated membrane proteins LAMP-1 and LAMP-2. Although the structure and function of ABC transporters were intensively analyzed in the past, the knowledge about accessory membrane embedded domains is limited.

Moving the Cellular Peptidome by Transporters.

Living matter is defined by metastability, implying a tightly balanced synthesis and turnover of cellular components. The first step of eukaryotic protein degradation via the ubiquitin-proteasome system (UPS) leads to peptides, which are subsequently degraded to single amino acids by an armada of proteases. A small fraction of peptides, however, escapes further cytosolic destruction and is transported by ATP-binding cassette (ABC) transporters into the endoplasmic reticulum (ER) and lysosomes.

The Design of New HIV-IN Tethered Bifunctional Inhibitors Using Multiple Microdomain Targeted Docking.

Currently, used antiretroviral HIV therapy drugs exclusively target critical groups in the enzymes essential for the viral life cycle. Increased mutagenesis of their genes changes these viral enzymes, which once mutated can evade therapeutic targeting, effects which confer drug resistance. To circumvent this, our review addresses a strategy to design and derive HIV-Integrase (HIV-IN) inhibitors which simultaneously target two IN functional domains, rendering it inactive even if the enzyme accumulates many mutations.

Structural characterization of the intrinsically disordered domain of Mycobacterium tuberculosis protein tyrosine kinase A.

Although intrinsically disordered proteins or protein domains (IDPs or IDD) are less abundant in bacteria than in eukaryotes, their presence in pathogenic bacterial proteins is important for protein-protein interactions. The protein tyrosine kinase A (PtkA) from Mycobacterium tuberculosis possesses an 80-residue disordered region (IDD ) of unknown function, located N-terminally to the well-folded kinase core domain. Here, we characterize the conformation of IDD under varying biophysical conditions and phosphorylation using NMR-spectroscopy.

Crystal structure and mechanistic basis of a functional homolog of the antigen transporter TAP.

ABC transporters form one of the largest protein superfamilies in all domains of life, catalyzing the movement of diverse substrates across membranes. In this key position, ABC transporters can mediate multidrug resistance in cancer therapy and their dysfunction is linked to various diseases. Here, we describe the 2.

Antigenic Peptide Recognition on the Human ABC Transporter TAP Resolved by DNP-Enhanced Solid-State NMR Spectroscopy.

The human transporter associated with antigen processing (TAP) is a 150 kDa heterodimeric ABC transport complex that selects peptides for export into the endoplasmic reticulum and subsequent loading onto major histocompatibility complex class I molecules to trigger adaptive immune responses against virally or malignantly transformed cells. To date, no atomic-resolution information on peptide-TAP interactions has been obtained, hampering a mechanistic understanding of the early steps of substrate translocation catalyzed by TAP. Here, we developed a mild method to concentrate an unstable membrane protein complex and combined this effort with dynamic nuclear polarization enhanced magic angle spinning solid-state NMR to study this challenging membrane protein-substrate complex.

Molecular characterization of methanogenic N(5)-methyl-tetrahydromethanopterin: Coenzyme M methyltransferase.

Methanogenic archaea share one ion gradient forming reaction in their energy metabolism catalyzed by the membrane-spanning multisubunit complex N(5)-methyl-tetrahydromethanopterin: coenzyme M methyltransferase (MtrABCDEFGH or simply Mtr). In this reaction the methyl group transfer from methyl-tetrahydromethanopterin to coenzyme M mediated by cobalamin is coupled with the vectorial translocation of Na(+) across the cytoplasmic membrane. No detailed structural and mechanistic data are reported about this process.

Interferon Alpha Signalling and Its Relevance for the Upregulatory Effect of Transporter Proteins Associated with Antigen Processing (TAP) in Patients with Malignant Melanoma.

Introduction: Interferon alpha (IFNα) is routinely used in the clinical practice for adjuvant systemic melanoma therapy. Understanding the molecular mechanism of IFNα effects and prediction of response in the IFNα therapy regime allows initiation and continuation of IFNα treatment for responder and exclusion of non-responder to avoid therapy inefficacy and side-effects. The transporter protein associated with antigen processing-1 (TAP1) is part of the MHC class I peptide-loading complex, and important for antigen presentation in tumor and antigen presenting cells.

Assembly of the MHC I peptide-loading complex determined by a conserved ionic lock-switch.

Salt bridges in lipid bilayers play a decisive role in the dynamic assembly and downstream signaling of the natural killer and T-cell receptors. Here, we describe the identification of an inter-subunit salt bridge in the membrane within yet another key component of the immune system, the peptide-loading complex (PLC). The PLC regulates cell surface presentation of self-antigens and antigenic peptides via molecules of the major histocompatibility complex class I.

Structure and Biophysical Characterization of the S-Adenosylmethionine-dependent O-Methyltransferase PaMTH1, a Putative Enzyme Accumulating during Senescence of Podospora anserina.

Low levels of reactive oxygen species (ROS) act as important signaling molecules, but in excess they can damage biomolecules. ROS regulation is therefore of key importance. Several polyphenols in general and flavonoids in particular have the potential to generate hydroxyl radicals, the most hazardous among all ROS.

An extended combinatorial 15N, 13Cα, and 13C' labeling approach to protein backbone resonance assignment.

Solution NMR studies of α-helical membrane proteins are often complicated by severe spectral crowding. In addition, hydrophobic environments like detergent micelles, isotropic bicelles or nanodiscs lead to considerably reduced molecular tumbling rates which translates into line-broadening and low sensitivity. Both difficulties can be addressed by selective isotope labeling methods.

Team work at its best – TAPL and its two domains.

The transporter associated with antigen processing (TAPL, ABCB9) is a homodimeric ABC transporter, shuttling cytosolic polypeptides into the lumen of lysosomes energized by ATP hydrolysis. Here we give a short overview of the superfamily of ABC transporters and summarize the current state of knowledge on TAPL in detail. The architecture of TAPL and its substrate specificity are described and we discuss the function of an extra N-terminal transmembrane domain, called TMD0, in respect of subcellular targeting and interaction with proteins, contributing to long-term stability.

Single liposome analysis of peptide translocation by the ABC transporter TAPL.

ATP-binding cassette (ABC) transporters use ATP to drive solute transport across biological membranes. Members of this superfamily have crucial roles in cell physiology, and some of the transporters are linked to severe diseases. However, understanding of the transport mechanism, especially of human ABC exporters, is scarce.

Modified vaccinia virus Ankara-infected dendritic cells present CD4+ T-cell epitopes by endogenous major histocompatibility complex class II presentation pathways.

Unlabelled: CD4(+) T lymphocytes play a central role in the immune system and mediate their function after recognition of their respective antigens presented on major histocompatibility complex II (MHCII) molecules on antigen-presenting cells (APCs). Conventionally, phagocytosed antigens are loaded on MHCII for stimulation of CD4(+) T cells. Certain epitopes, however, can be processed directly from intracellular antigens and are presented on MHCII (endogenous MHCII presentation).

Time-shared experiments for efficient assignment of triple-selectively labeled proteins.

Combinatorial triple-selective labeling facilitates the NMR assignment process for proteins that are subject to signal overlap and insufficient signal-to-noise in standard triple-resonance experiments. Aiming at maximum amino-acid type and sequence-specific information, the method represents a trade-off between the number of selectively labeled samples that have to be prepared and the number of spectra to be recorded per sample. In order to address the demand of long measurement times, we here propose pulse sequences in which individual phase-shifted transients are stored separately and recombined later to produce several 2D HN(CX) type spectra that are usually acquired sequentially.

Mechanistic determinants of the directionality and energetics of active export by a heterodimeric ABC transporter.

The ATP-binding cassette (ABC) transporter associated with antigen processing (TAP) participates in immune surveillance by moving proteasomal products into the endoplasmic reticulum (ER) lumen for major histocompatibility complex class I loading and cell surface presentation to cytotoxic T cells. Here we delineate the mechanistic basis for antigen translocation. Notably, TAP works as a molecular diode, translocating peptide substrates against the gradient in a strict unidirectional way.