A perspective on the structural studies of inner membrane electrochemical potential-driven transporters

Electrochemical potential-driven transporters represent a vast array of proteins with varied substrate specificities. While diverse in size and substrate specificity, they are all driven by electrochemical potentials. Over the past five years there have been increasing numbers of X-ray structures reported for this family of transporters. Structural information is available for five subfamilies of electrochemical potential-driven transporters. No structural information exists for the remaining 91 subfamilies. In this review, the various subfamilies of electrochemical potential-driven transporters are discussed. The seven reported structures for the electrochemical potential-driven transporters and the methods for their crystallization are also presented. With a few exceptions, overall crystallization trends have been very similar for the transporters despite their differences in substrate specificity and topology. Also discussed is why the structural studies on these transporters were successful while others are not as fruitful. With the plethora of transporters with unknown structures, this review provides incentive for crystallization of transporters in the remaining subfamilies for which no structural information exists.     

Principles of Alternating Access in Multidrug and Toxin Extrusion (MATE) Transporters

The multidrug and toxin extrusion (MATE) transporters catalyze active efflux of a broad range of chemically- and structurally-diverse compounds including antimicrobials and chemotherapeutics, thus contributing to multidrug resistance in pathogenic bacteria and cancers. Multiple methodological approaches have been taken to investigate the structural basis of energy transduction and substrate translocation in MATE transporters. Crystal structures representing members from all three MATE subfamilies have been interpreted within the context of an alternating access mechanism that postulates occupation of distinct structural intermediates in a conformational cycle powered by electrochemical ion gradients. Here we review the structural biology of MATE transporters, integrating the crystallographic models with biophysical and computational studies to define the molecular determinants that shape the transport energy landscape. This holistic analysis highlights both shared and disparate structural and functional features within the MATE family, which underpin an emerging theme of mechanistic diversity within the framework of a conserved structural scaffold.     

ABC transporter architecture and mechanism: implications from the crystal structures of BtuCD and BtuF

ABC transporters are ubiquitous membrane proteins that facilitate unidirectional substrate translocation across the lipid bilayer. Over the past five years, new crystal structures have advanced our understanding of how ABC transporters couple adenosine triphosphate (ATP) hydrolysis to substrate transport. In the following, we will briefly review the results of these structural investigations and outline their mechanistic implications.     

Structure determination of channel and transport proteins by high-resolution microscopy techniques

High-resolution microscopy techniques provide a plethora of information on biological structures from the cellular level down to the molecular level. In this review, we present the unique capabilities of transmission electron and atomic force microscopy to assess the structure, oligomeric state, function and dynamics of channel and transport proteins in their native environment, the lipid bilayer. Most importantly, membrane proteins can be visualized in the frozen-hydrated state and in buffer solution by cryo-transmission electron and atomic force microscopy, respectively. We also illustrate the potential of the scintillation proximity assay to study substrate binding of detergent-solubilized transporters prior to crystallization and structural characterization.     

Eukaryotic major facilitator superfamily transporter modeling based on the prokaryotic GlpT crystal structure

The major facilitator superfamily (MFS) of transporters represents the largest family of secondary active transporters and has a diverse range of substrates. With structural information for four MFS transporters, we can see a strong structural commonality suggesting, as predicted, a common architecture for MFS transporters. The rate for crystal structure determination of MFS transporters is slow, making modeling of both prokaryotic and eukaryotic transporters more enticing. In this review, models of eukaryotic transporters Glut1, G6PT, OCT1, OCT2 and Pho84, based on the crystal structures of the prokaryotic GlpT, based on the crystal structure of LacY are discussed. The techniques used to generate the different models are compared. In addition, the validity of these models and the strategy of using prokaryotic crystal structures to model eukaryotic proteins are discussed. For comparison, E. coli GlpT was modeled based on the E. coli LacY structure and compared to the crystal structure of GlpT demonstrating that experimental evidence is essential for accurate modeling of membrane proteins.     

Functional discrimination of membrane proteins using machine learning techniques

Background  

Discriminating membrane proteins based on their functions is an important task in genome annotation. In this work, we have analyzed the characteristic features of amino acid residues in membrane proteins that perform major functions, such as channels/pores, electrochemical potential-driven transporters and primary active transporters.     

Eukaryotic major facilitator superfamily transporter modeling based on the prokaryotic GlpT crystal structure (Review)

The major facilitator superfamily (MFS) of transporters represents the largest family of secondary active transporters and has a diverse range of substrates. With structural information for four MFS transporters, we can see a strong structural commonality suggesting, as predicted, a common architecture for MFS transporters. The rate for crystal structure determination of MFS transporters is slow, making modeling of both prokaryotic and eukaryotic transporters more enticing. In this review, models of eukaryotic transporters Glut1, G6PT, OCT1, OCT2 and Pho84, based on the crystal structures of the prokaryotic GlpT, based on the crystal structure of LacY are discussed. The techniques used to generate the different models are compared. In addition, the validity of these models and the strategy of using prokaryotic crystal structures to model eukaryotic proteins are discussed. For comparison, E. coli GlpT was modeled based on the E. coli LacY structure and compared to the crystal structure of GlpT demonstrating that experimental evidence is essential for accurate modeling of membrane proteins.     

Structure determination of secondary transport proteins by electron crystallography: two-dimensional crystallization of the betaine uptake system BetP

Structure determination at high resolution is still a challenge for membrane proteins in general, but in particular for secondary transporters due to their highly dynamic nature. X-ray structures of ten secondary transporters have recently been determined, but a thorough understanding of transport mechanisms necessitates structures at different functional states. Electron cryo-microscopy of two-dimensional (2D) crystals offers an alternative to obtain structural information at intermediate resolution. Electron crystallography is a sophisticated way to study proteins in a natural membrane environment and to track conformational changes in situ. Furthermore, basic interactions between protein and lipids can be investigated. Projection and 3-dimensional maps of six secondary transporters from different families have been determined by electron crystallography of 2D crystals at a resolution of 8 A and better. In this review, we give an overview about the principles of 2D crystallization, in particular of secondary transporters, and summarize the important steps successfully applied to establish and improve the 2D crystallization of the high-affinity glycine betaine uptake system from Corynebacterium glutamicum, BetP.     

Differences in the substrate specificities and active-site structures of two α-L-fucosidases (glycoside hydrolase family 29) from Bacteroides thetaiotaomicron

Recent studies suggest that α-L-fucosidases of glycoside hydrolase family 29 can be divided into two subfamilies based on substrate specificity and phylogenetic clustering. To explore the validity of this classification, we enzymatically characterized two structure-solved α-L-fucosidases representing the respective subfamilies. Differences in substrate specificities are discussed in relation to differences in active-site structures between the two enzymes.     

Structures of multidrug and toxic compound extrusion transporters and their mechanistic implications

Multidrug resistance poses grand challenges to the effective treatment of infectious diseases and cancers. Integral membrane proteins from the multidrug and toxic compound extrusion (MATE) family contribute to multidrug resistance by exporting a wide variety of therapeutic drugs across cell membranes. MATE proteins are conserved from bacteria to humans and can be categorized into the NorM, DinF and eukaryotic subfamilies. MATE transporters hold great appeal as potential therapeutic targets for curbing multidrug resistance, yet their transport mechanism remains elusive. During the past 5 years, X-ray structures of 4 NorM and DinF transporters have been reported and guided biochemical studies to reveal how MATE transporters extrude different drugs. Such advances, although substantial, have yet to be discussed collectively. Herein I review these structures and the unprecedented mechanistic insights that have been garnered from those structure-inspired studies, as well as lay out the outstanding questions that present exciting opportunities for future work.     

Thermodynamic secrets of multidrug resistance: A new take on transport mechanisms of secondary active antiporters

Multidrug resistance (MDR) presents a growing challenge to global public health. Drug extrusion transporters play a critical part in MDR; thus, their mechanisms of substrate recognition are being studied in great detail. In this work, we review common structural features of key transporters involved in MDR. Based on our membrane potential‐driving hypothesis, we propose a general energy‐coupling mechanism for secondary‐active antiporters. This putative mechanism provides a common framework for understanding poly‐specificity of most—if not all—MDR transporters.     

Substrate specificity of protein kinases and computational prediction of substrates

To ensure signalling fidelity, kinases must act only on a defined subset of cellular targets. Appreciating the basis for this substrate specificity is essential for understanding the role of an individual protein kinase in a particular cellular process. The specificity in the cell is determined by a combination of "peptide specificity" of the kinase (the molecular recognition of the sequence surrounding the phosphorylation site), substrate recruitment and phosphatase activity. Peptide specificity plays a crucial role and depends on the complementarity between the kinase and the substrate and therefore on their three-dimensional structures. Methods for experimental identification of kinase substrates and characterization of specificity are expensive and laborious, therefore, computational approaches are being developed to reduce the amount of experimental work required in substrate identification. We discuss the structural basis of substrate specificity of protein kinases and review the experimental and computational methods used to obtain specificity information.     

Differences in the Substrate Specificities and Active-Site Structures of Two α-L-Fucosidases (Glycoside Hydrolase Family 29) from Bacteroides thetaiotaomicron

Recent studies suggest that α-L-fucosidases of glycoside hydrolase family 29 can be divided into two subfamilies based on substrate specificity and phylogenetic clustering. To explore the validity of this classification, we enzymatically characterized two structure-solved α-L-fucosidases representing the respective subfamilies. Differences in substrate specificities are discussed in relation to differences in active-site structures between the two enzymes.     

Molecular characteristics of transporters of C<Subscript>4</Subscript>-dicarboxylates and mechanism of translocation

An important role in cell metabolism is played by transport of C₄-dicarboxylates (C₄-DCB). Specifically, they are intermediates of the citrate cycle. Transport of succinate across the mitochondrial membrane provides correlation between metabolism in peroxysomes and in mitochondria. There is known transport of C₄-DCB across all kinds of energy-transforming membranes of the animal, plant, fungal, and bacterial cells. This review summarizes molecular characteristics of the C₄-DCB transporters. Of particular interest are primary structures for the transporters with the known kinetic mechanism and kinetic transport parameters. For each studied group of organisms, the number of transmembrane segments in the carried molecule or the character of specificity does not correlate with the certain transport mechanism—antiport, symport with proton or symport with cation. The review describes perspective methodical approaches allowing association of peculiarities of structure with transport mechanism for individual transporters, preparation of functional hybrid transporters—“protein chimeras,” scanning of transporter transmembrane segments with aid of essential acids, probing of the transporter active center with aid of alkyl and acyl substrate derivatives used to obtain the “lipophilic profile” of the channel of the C₄-DCB transporter. It is recommended to use these approaches to one transporter that has small sizes and large substrate specificity.     

The human mitochondrial transport protein family: identification and protein regions significant for transport function and substrate specificity

Protein sequence similarities and predicted structures identified 75 mitochondrial transport proteins (37 subfamilies) from among the 28,994 human RefSeq (NCBI) protein sequences. All, except two, have an E-value of less than 4e--05 with respect to the structure of the single subunit bovine ADP/ATP carrier/carboxyatractyloside complex (bAAC/CAT) (mGenThreader program). The two 30-kDa exceptions have E-values of 0.003 and 0.005. 21 have been functionally identified and belong to 14 subfamilies. A subset of subfamilies with sequence similarities for each of 12 different protein regions was identified. Many of the 12 protein regions for each tested protein yielded different size subsets. The sum of subfamilies in the 12 subsets was lowest for the phosphate transport protein (PTP) and highest for aralar 1. Transmembrane sequences are most unique. Sequence similarities are highest near the membrane center and matrix. They are highest for the region of transmembrane helices H1, H2 and connecting matrix loop 12 and smallest for transmembrane helices H3, H4 and loop 34. These sequence similarities and the predicted high similarities to the bAAC/CAT structure point to common structural/functional elements that could include subunit/subunit contact sites as they have been identified for PTP and AAC. The four residues protein segment (SerLysGlnIle) of loop 12 is the only segment projecting into the center of the funnel-like structure of the bAAC/CAT. It is present in its entirety only in the AACs and with some replacements in the large Ca2+-modulated aspartate/glutamate transporters. Other transporters have deletions and replacements in this region of loop 12. This protein segment with its central location and variation in size and composition likely contributes to the substrate specificity of the transporters.     

Insight in eukaryotic ABC transporter function by mutation analysis

With regard to structure-function relations of ATP-binding cassette (ABC) transporters several intriguing questions are in the spotlight of active research: Why do functional ABC transporters possess two ATP binding and hydrolysis domains together with two ABC signatures and to what extent are the individual nucleotide-binding domains independent or interacting? Where is the substrate-binding site and how is ATP hydrolysis functionally coupled to the transport process itself? Although much progress has been made in the elucidation of the three-dimensional structures of ABC transporters in the last years by several crystallographic studies including novel models for the nucleotide hydrolysis and translocation catalysis, site-directed mutagenesis as well as the identification of natural mutations is still a major tool to evaluate effects of individual amino acids on the overall function of ABC transporters. Apart from alterations in characteristic sequence such as Walker A, Walker B and the ABC signature other parts of ABC proteins were subject to detailed mutagenesis studies including the substrate-binding site or the regulatory domain of CFTR. In this review, we will give a detailed overview of the mutation analysis reported for selected ABC transporters of the ABCB and ABCC subfamilies, namely HsCFTR/ABCC7, HsSUR/ABCC8,9, HsMRP1/ABCC1, HsMRP2/ABCC2, ScYCF1 and P-glycoprotein (Pgp)/MDR1/ABCB1 and their effects on the function of each protein.     

The human mitochondrial transport protein family: Identification and protein regions significant for transport function and substrate specificity

Protein sequence similarities and predicted structures identified 75 mitochondrial transport proteins (37 subfamilies) from among the 28,994 human RefSeq (NCBI) protein sequences. All, except two, have an E-value of less than 4e−05 with respect to the structure of the single subunit bovine ADP/ATP carrier/carboxyatractyloside complex (bAAC/CAT) (mGenThreader program). The two 30-kDa exceptions have E-values of 0.003 and 0.005. 21 have been functionally identified and belong to 14 subfamilies. A subset of subfamilies with sequence similarities for each of 12 different protein regions was identified. Many of the 12 protein regions for each tested protein yielded different size subsets. The sum of subfamilies in the 12 subsets was lowest for the phosphate transport protein (PTP) and highest for aralar 1. Transmembrane sequences are most unique. Sequence similarities are highest near the membrane center and matrix. They are highest for the region of transmembrane helices H1, H2 and connecting matrix loop 12 and smallest for transmembrane helices H3, H4 and loop 34. These sequence similarities and the predicted high similarities to the bAAC/CAT structure point to common structural/functional elements that could include subunit/subunit contact sites as they have been identified for PTP and AAC. The four residues protein segment (SerLysGlnIle) of loop 12 is the only segment projecting into the center of the funnel-like structure of the bAAC/CAT. It is present in its entirety only in the AACs and with some replacements in the large Ca2+-modulated aspartate/glutamate transporters. Other transporters have deletions and replacements in this region of loop 12. This protein segment with its central location and variation in size and composition likely contributes to the substrate specificity of the transporters.     

Structural Features of the Glutamate Transporter Family

Neuronal and glial glutamate transporters remove the excitatory neurotransmitter glutamate from the synaptic cleft and thus prevent neurotoxicity. The proteins belong to a large and widespread family of secondary transporters, including bacterial glutamate, serine, and C₄-dicarboxylate transporters; mammalian neutral-amino-acid transporters; and an increasing number of bacterial, archaeal, and eukaryotic proteins that have not yet been functionally characterized. Sixty members of the glutamate transporter family were found in the databases on the basis of sequence homology. The amino acid sequences of the carriers have diverged enormously. Homology between the members of the family is most apparent in a stretch of approximately 150 residues in the C-terminal part of the proteins. This region contains four reasonably well-conserved sequence motifs, all of which have been suggested to be part of the translocation pore or substrate binding site. Phylogenetic analysis of the C-terminal stretch revealed the presence of five subfamilies with characterized members: (i) the eukaryotic glutamate transporters, (ii) the bacterial glutamate transporters, (iii) the eukaryotic neutral-amino-acid transporters, (iv) the bacterial C₄-dicarboxylate transporters, and (v) the bacterial serine transporters. A number of other subfamilies that do not contain characterized members have been defined. In contrast to their amino acid sequences, the hydropathy profiles of the members of the family are extremely well conserved. Analysis of the hydropathy profiles has suggested that the glutamate transporters have a global structure that is unique among secondary transporters. Experimentally, the unique structure of the transporters was recently confirmed by membrane topology studies. Although there is still controversy about part of the topology, the most likely model predicts the presence of eight membrane-spanning α-helices and a loop-pore structure which is unique among secondary transporters but may resemble loop-pores found in ion channels. A second distinctive structural feature is the presence of a highly amphipathic membrane-spanning helix that provides a hydrophilic path through the membrane. Recent data from analysis of site-directed mutants and studies on the mechanism and pharmacology of the transporters are discussed in relation to the structural model.     

Structural features of the glutamate transporter family.

Neuronal and glial glutamate transporters remove the excitatory neurotransmitter glutamate from the synaptic cleft and thus prevent neurotoxicity. The proteins belong to a large and widespread family of secondary transporters, including bacterial glutamate, serine, and C4-dicarboxylate transporters; mammalian neutral-amino-acid transporters; and an increasing number of bacterial, archaeal, and eukaryotic proteins that have not yet been functionally characterized. Sixty members of the glutamate transporter family were found in the databases on the basis of sequence homology. The amino acid sequences of the carriers have diverged enormously. Homology between the members of the family is most apparent in a stretch of approximately 150 residues in the C-terminal part of the proteins. This region contains four reasonably well-conserved sequence motifs, all of which have been suggested to be part of the translocation pore or substrate binding site. Phylogenetic analysis of the C-terminal stretch revealed the presence of five subfamilies with characterized members: (i) the eukaryotic glutamate transporters, (ii) the bacterial glutamate transporters, (iii) the eukaryotic neutral-amino-acid transporters, (iv) the bacterial C4-dicarboxylate transporters, and (v) the bacterial serine transporters. A number of other subfamilies that do not contain characterized members have been defined. In contrast to their amino acid sequences, the hydropathy profiles of the members of the family are extremely well conserved. Analysis of the hydropathy profiles has suggested that the glutamate transporters have a global structure that is unique among secondary transporters. Experimentally, the unique structure of the transporters was recently confirmed by membrane topology studies. Although there is still controversy about part of the topology, the most likely model predicts the presence of eight membrane-spanning alpha-helices and a loop-pore structure which is unique among secondary transporters but may resemble loop-pores found in ion channels. A second distinctive structural feature is the presence of a highly amphipathic membrane-spanning helix that provides a hydrophilic path through the membrane. Recent data from analysis of site-directed mutants and studies on the mechanism and pharmacology of the transporters are discussed in relation to the structural model.     

Classification of transporters using efficient radial basis function networks with position‐specific scoring matrices and biochemical properties

Transporters are proteins that are involved in the movement of ions or molecules across biological membranes. Transporters are generally classified into channels/pores, electrochemical transporters, and active transporters. Discriminating the specific class of transporters and their subfamilies are essential tasks in computational biology for the advancement of structural and functional genomics. We have systematically analyzed the amino acid composition, residue pair preference and amino acid properties in six different families of transporters. Utilizing the information, we have developed a radial basis function (RBF) network method based on profiles obtained with position specific scoring matrices for discriminating transporters belonging to three different classes and six families. Our method showed a fivefold cross validation accuracy of 76%, 73%, and 69% for discriminating transporters and nontransporters, three different classes and six different families of transporters, respectively. Further, the method was tested with independent datasets, which showed similar level of accuracy. A web server has been developed for discriminating transporters based on three classes and six families, and it is available at http://rbf.bioinfo.tw/～sachen/tcrbf.html . We suggest that our method could be effectively used to identify transporters and discriminating them into different classes and families. Proteins 2010;. © 2010 Wiley‐Liss, Inc.