How to become a successful scientist: the 2022 CSMB Arthur Wynne Gold Medal Lecture.

There are many pathways to success. Mine followed a traditional one to an academic faculty position, but this pathway is not the one most life sciences PhD graduates will follow today. We have all had time during the COVID-19 pandemic to reflect on our personal pathway-where we are and where we are going.

Organization and Dynamics of the Red Blood Cell Band 3 Anion Exchanger SLC4A1: Insights From Molecular Dynamics Simulations.

Molecular dynamics (MD) simulations have provided new insights into the organization and dynamics of the red blood cell Band 3 anion exchanger (AE1, SLC4A1). Band 3, like many solute carriers, works by an alternating access mode of transport where the protein rapidly (10/s) changes its conformation between outward and inward-facing states a transient occluded anion-bound intermediate. While structural studies of membrane proteins usually reveal valuable structural information, these studies provide a static view often in the presence of detergents.

Molecular Simulations of Intact Anion Exchanger 1 Reveal Specific Domain and Lipid Interactions.

Anion exchanger 1 (AE1) is responsible for the exchange of bicarbonate and chloride across the erythrocyte plasma membrane. Human AE1 consists of a cytoplasmic and a membrane domain joined by a 33-residue flexible linker. Crystal structures of the individual domains have been determined, but the intact AE1 structure remains elusive.

Role of N-glycosylation in the expression of human SLC26A2 and A3 anion transport membrane glycoproteins .

The human solute carrier 26 (SLC26) gene family of anion transporters consists of 10 members (-, being a pseudogene) that encode membrane glycoproteins with 14 transmembrane segments and a C-terminal cytoplasmic sulfate transporter anti-sigma antagonist domain. Thus far, mutations in eight members of the family (-, , and ) have been linked to diseases in humans. Our goal is to characterize the role of N-glycosylation and the effect of mutations in SLC26A2 and A3 proteins on their functional expression in transfected HEK-293 cells.

Interaction of the human erythrocyte Band 3 anion exchanger 1 (AE1, SLC4A1) with lipids and glycophorin A: Molecular organization of the Wright (Wr) blood group antigen.

The Band 3 (AE1, SLC4A1) membrane protein is found in red blood cells and in kidney where it functions as an electro-neutral chloride/bicarbonate exchanger. In this study, we have used molecular dynamics simulations to provide the first realistic model of the dimeric membrane domain of human Band 3 in an asymmetric lipid bilayer containing a full complement of phospholipids, including phosphatidylinositol 4,5-bisphosphate (PIP2) and cholesterol, and its partner membrane protein Glycophorin A (GPA). The simulations show that the annular layer in the inner leaflet surrounding Band 3 was enriched in phosphatidylserine and PIP2 molecules.

Structural biology of solute carrier (SLC) membrane transport proteins.

The human solute carriers (SLCs) comprise over 400 different transporters, organized into 65 families ( http://slc.bioparadigms.org/ ) based on their sequence homology and transport function.

Band 3 function and dysfunction in a structural context.

Purpose Of Review: Current research on the human band 3 glycoprotein, the red cell chloride/bicarbonate anion exchanger (AE1), is highlighted and placed within a structural context.

Recent Findings: The determination of the crystal structure of the membrane domain of human band 3, the founding member of the solute carrier 4 (SLC4) family of bicarbonate transporters, is a major breakthrough toward understanding the mechanism of action of this membrane transport protein, its interaction with partner proteins, and how mutations linked to disease affect its ability to fold and function.

Summary: Band 3 contains 14 transmembrane segments arranged in a 7+7 transmembrane inverted repeat topology common to all members of the SLC4 family and the unrelated SLC26 anion transporter family.

Molecular analysis of human solute carrier SLC26 anion transporter disease-causing mutations using 3-dimensional homology modeling.

The availability of the first crystal structure of a bacterial member (SLC26Dg) of the solute carrier SLC26 family of anion transporters has allowed us to create 3-dimensional models of all 10 human members (SLC26A1-A11, A10 being a pseudogene) of these membrane proteins using the Phyre2 bioinformatic tool. The homology modeling predicted that the SLC26 human proteins, like the SLC26Dg template, all consist of 14 transmembrane segments (TM) arranged in a 7+7 inverted topology with the amino-termini of two half-helices (TM3 and 10) facing each other in the centre of the protein to create the anion-binding site, linked to a C-terminal cytosolic sulfate transporter anti-sigma factor antagonist (STAS) domain. A plethora of human diseases are associated with mutations in the genes encoding human SLC26 transporters, including chondrodysplasias with varying severity in SLC26A2 (~50 mutations, 27 point mutations), congenital chloride-losing diarrhea in SLC26A3 (~70 mutations, 31 point mutations) and Pendred Syndrome or deafness autosomal recessive type 4 in SLC26A4 (~500 mutations, 203 point mutations).

N-linked glycosylation of a subunit isoforms is critical for vertebrate vacuolar H -ATPase (V-ATPase) biosynthesis.

The a subunit of the V membrane-integrated sector of human V-ATPase has four isoforms, a1-a4, with diverse and crucial functions in health and disease. They are encoded by four conserved paralogous genes, and their vertebrate orthologs have positionally conserved N-glycosylation sequons within the second extracellular loop, EL2, of the a subunit membrane domain. Previously, we have shown directly that the predicted sequon for the a4 isoform is indeed N-glycosylated.

Effect of the Southeast Asian Ovalocytosis Deletion on the Conformational Dynamics of Signal-Anchor Transmembrane Segment 1 of Red Cell Anion Exchanger 1 (AE1, Band 3, or SLC4A1).

The first transmembrane (TM1) helix in the red cell anion exchanger (AE1, Band 3, or SLC4A1) acts as an internal signal anchor that binds the signal recognition particle and directs the nascent polypeptide chain to the endoplasmic reticulum (ER) membrane where it moves from the translocon laterally into the lipid bilayer. The sequence N-terminal to TM1 forms an amphipathic helix that lies at the membrane interface and is connected to TM1 by a bend at Pro403. Southeast Asian ovalocytosis (SAO) is a red cell abnormality caused by a nine-amino acid deletion (Ala400-Ala408) at the N-terminus of TM1.

Band 3, the human red cell chloride/bicarbonate anion exchanger (AE1, SLC4A1), in a structural context.

The crystal structure of the dimeric membrane domain of human Band 3(1), the red cell chloride/bicarbonate anion exchanger 1 (AE1, SLC4A1), provides a structural context for over four decades of studies into this historic and important membrane glycoprotein. In this review, we highlight the key structural features responsible for anion binding and translocation and have integrated the following topological markers within the Band 3 structure: blood group antigens, N-glycosylation site, protease cleavage sites, inhibitor and chemical labeling sites, and the results of scanning cysteine and N-glycosylation mutagenesis. Locations of mutations linked to human disease, including those responsible for Southeast Asian ovalocytosis, hereditary stomatocytosis, hereditary spherocytosis, and distal renal tubular acidosis, provide molecular insights into their effect on Band 3 folding.

Effect of SLC26 anion transporter disease-causing mutations on the stability of the homologous STAS domain of E. coli DauA (YchM).

The human solute carrier 26 (SLC26) family of anion transporters consists of ten members that are found in various organs in the body including the stomach, intestine, kidney, thyroid and ear where they transport anions including bicarbonate, chloride and sulfate, typically in an exchange mode. Mutations in these genes cause a plethora of diseases such as diastrophic dysplasia affecting sulfate uptake into chondrocytes (SLC26A2), congenital chloride-losing diarrhoea (SLC26A3) affecting chloride secretion in the intestine and Pendred's syndrome (SLC26A4) resulting in hearing loss. To understand how these mutations affect the structures of the SLC26 membrane proteins and their ability to function properly, 12 human disease-causing mutants from SLC26A2, SLC26A3 and SLC26A4 were introduced into the equivalent sites of the sulfate transporter anti-sigma factor antagonist (STAS) domain of a bacterial homologue SLC26 protein DauA (YchM).

Molecular dynamics simulations of the bacterial UraA H+-uracil symporter in lipid bilayers reveal a closed state and a selective interaction with cardiolipin.

The Escherichia coli UraA H+-uracil symporter is a member of the nucleobase/ascorbate transporter (NAT) family of proteins, and is responsible for the proton-driven uptake of uracil. Multiscale molecular dynamics simulations of the UraA symporter in phospholipid bilayers consisting of: 1) 1-palmitoyl 2-oleoyl-phosphatidylcholine (POPC); 2) 1-palmitoyl 2-oleoyl-phosphatidylethanolamine (POPE); and 3) a mixture of 75% POPE, 20% 1-palmitoyl 2-oleoyl-phosphatidylglycerol (POPG); and 5% 1-palmitoyl 2-oleoyl-diphosphatidylglycerol/cardiolipin (CL) to mimic the lipid composition of the bacterial inner membrane, were performed using the MARTINI coarse-grained force field to self-assemble lipids around the crystal structure of this membrane transport protein, followed by atomistic simulations. The overall fold of the protein in lipid bilayers remained similar to the crystal structure in detergent on the timescale of our simulations.

Differential roles of tryptophan residues in the functional expression of human anion exchanger 1 (AE1, Band 3, SLC4A1).

Anion exchanger 1 (AE1) is a 95 kDa glycoprotein that facilitates Cl(-)=HCO(-)(3) exchange across the erythrocyte plasma membrane. This transport activity resides in the 52 kDa C-terminal membrane domain (Gly(361)-Val(911)) predicted to span the membrane 14 times. To explore the role of tryptophan (Trp) residues in AE1 function, the seven endogenous Trp residues in the membrane domain were mutated individually to alanine (Ala) and phenylalanine (Phe).

Lessons from a red squirrel, mentors, and the pathway to success.

In this article I will review my personal career path starting with how a red squirrel got me interested in research, and the vital role that mentors played in my pathway to success - a pathway that taught me many lessons that I would like to share with the reader, particularly graduate students and post-doctoral fellows who are just starting down their own unique pathways.

Structure, function, and trafficking of SLC4 and SLC26 anion transporters.

The structure and function of the red cell anion exchanger 1 (AE1, Band 3, SLC4A1), the truncated kidney anion exchanger 1 (kAE1), and the other members of the SLC4 family of bicarbonate transporters are reviewed. Mutations in the AE1 gene cause human diseases like Southeast Asian ovalocytosis and hereditary spherocytosis in the red cell and distal renal tubular acidosis in the kidney. These mutations affect the folding, trafficking, and functional expression of these membrane glycoproteins.

N-glycosylation and topology of the human SLC26 family of anion transport membrane proteins.

The human solute carrier (SLC26) family of anion transporters consists of 10 members (SLCA1-11, SLCA10 being a pseudogene) that encode membrane proteins containing ~12 transmembrane (TM) segments with putative N-glycosylation sites (-NXS/T-) in extracellular loops and a COOH-terminal cytosolic STAS domain. All 10 members of the human SLC26 family, FLAG-tagged at the NH2 terminus, were transiently expressed in HEK-293 cells. While most proteins were observed to contain both high-mannose and complex oligosaccharides, SLC26A2 was mainly in the complex form, SLC26A4 in the high-mannose form, and SLC26A8 was not N-glycosylated.

A substrate access tunnel in the cytosolic domain is not an essential feature of the solute carrier 4 (SLC4) family of bicarbonate transporters.

Anion exchanger 1 (AE1; Band 3; SLC4A1) is the founding member of the solute carrier 4 (SLC4) family of bicarbonate transporters that includes chloride/bicarbonate AEs and Na(+)-bicarbonate co-transporters (NBCs). These membrane proteins consist of an amino-terminal cytosolic domain involved in protein interactions and a carboxyl-terminal membrane domain that carries out the transport function. Mutation of a conserved arginine residue (R298S) in the cytosolic domain of NBCe1 (SLC4A4) is linked to proximal renal tubular acidosis and results in impaired transport function, suggesting that the cytosolic domain plays a role in substrate permeation.

Aerosolized liposomal Amphotericin B: a potential prophylaxis of invasive pulmonary aspergillosis in immunocompromised patients.

Background: Aerosolized liposomal Amphotericin B may reduce the incidence of invasive pulmonary Aspergillosis in adults with chemotherapy-induced prolonged neutropenia with less nephrotoxicity. The breath-actuated AeroEclipse® BAN nebulizer is very efficient and minimizes environmental drug contamination since no aerosol is produced, unless the patient is inspiring through the device. Our aim is to develop an appropriate delivery system suitable for children that does not disrupt the liposomes due to the shear forces in nebulization.

Membrane transport metabolons.

In this review evidence from a wide variety of biological systems is presented for the genetic, functional, and likely physical association of membrane transporters and the enzymes that metabolize the transported substrates. This evidence supports the hypothesis that the dynamic association of transporters and enzymes creates functional membrane transport metabolons that channel substrates typically obtained from the extracellular compartment directly into their cellular metabolism. The immediate modification of substrates on the inner surface of the membrane prevents back-flux through facilitated transporters, increasing the efficiency of transport.

Report on the Bicarbonate Transport Satellite Meeting at the 53rd Annual Meeting of the Canadian Society of Biochemistry, Molecular and Cellular Biology.

The Bicarbonate Transport Meeting was held as a satellite meeting of the 53rd Annual Meeting of the Canadian Society of Biochemistry, Molecular and Cellular Biology (CSBMCB): Membrane Proteins in Health and Disease. The meeting covered the modern history of bicarbonate transporter proteins and brought together the major workers in the field. Ron Kopito recounted the story of the first determination of the amino acid sequence for a bicarbonate transporter, AE1/Band 3, 25 years earlier while working with Harvey Lodish at Harvard, while Tomohiro Yamaguchi and Teruhisa Hirai presented up-to-date data on AE1 structure obtained using electron crystallography.