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Copyright
by
Aditya D. Joshi
2008
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The Dissertation Committee for Aditya D. Joshi certifies that this is the approved version of the following dissertation:
STRUCTURAL AND FUNCTIONAL STUDIES OF Na+/DICARBOXYLATE COTRANSPORTERS
Committee:
Ana M. Pajor, Ph.D., SupervisorSteven C. King, Ph.D.Simon A. Lewis, Ph.D.Krishna Rajarathnam, Ph.D.Nancy K. Wills, Ph.D.
Dean, Graduate School
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STRUCTURAL AND FUNCTIONAL STUDIES OF Na+/DICARBOXYLATE COTRANSPORTERS
By
Aditya D. Joshi, M. Sc.
HIDDEN TEXT: Given first name, and previous academic degrees (B.A. or higher) B.A., B.S., etc. Your official name is the name which appears on your UT transcript.
Dissertation
Presented to the Faculty of the Graduate School of Biomedical Sciences
The University of Texas Medical Branch
in Partial Fulfillment
of the Requirements
for the Degree of
Doctor of Philosophy
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The University of Texas Medical Branch
August 2008
To my dearest family
ACKNOWLEDGEMENTS
From bottom of my heart, I would like to convey my sincere appreciation and gratitude to my extremely inspirational and supportive mentor, Dr. Ana M. Pajor for helping me throughout my Ph.D. studies. I am deeply thankful to her for providing me with the invaluable resources in the form of constant encouragement, advice, inspiration, compassion as well as thought provoking ideas, which helped me to complete my doctoral work. I truly believe she has been a wonderful mentor and I am indebted to her for all her support.
I would also like to express my sincere gratitude to Dr. Simon A. Lewis, for providing me insights on understanding physiological aspects of structure-function studies as well as his constant encouragement. I am thankful to Dr. Steven C. King for his advice on Transport Specificity Ratio experiments as well as his constant suggestions during different stages of this dissertation work. I am also grateful to Dr. Nancy K. Wills and Dr. Krishna Rajarathnam for providing me with invaluable advice on experimental design, data interpretation and useful discussions.
I really like to appreciate my colleagues and friends Dr. Jason A. Hall, Dr. Jittima Weerachayaphorn, Dr. Naomi Oshiro, Donovan E. Randolph, Jamie R. Lewis, Dr. Alieen K. Ritchie, Cynthia Cheatham, Aditya Hindupur, Rohit Jangra, Abhisek Mukharjee, and Dr. Lokesh Rao for being helpful and supportive. I would especially like to thank Kathleen M. Randolph not only for her scientific and technical inputs, but also for her joyful and positive encouragement.
I sincerely thank Dr. Werner Braun for giving me an opportunity to work in the Sealy Center for Computational Biology and his constant suggestions regarding the computational modeling aspects of this project. I also thank Dr. Surendra Negi for helping me with NaDC1 computational modeling.
I would like to express my deepest appreciation to my parents, Sunanda and Dilip Joshi, my brother Ojas, sister-in-law, Ketki and my adorable niece, Saniya for inspiring me to put in my best efforts in carrying out this work and their constant encouragement.
I feel that the words are not enough to express my appreciation to my lovely wife, Trupti for being supportive and her efforts to help me make it possible.
STRUCTURAL AND FUNCTIONAL STUDIES OF Na+/DICARBOXYLATE COTRANSPORTERS
Publication No._____________
Aditya D. Joshi, M. Sc., Ph. D.
The University of Texas Medical Branch, June 2008
Supervisor: Ana M. Pajor
The Na+/dicarboxylate cotransporter (NaDC1) is found on the apical membrane of the kidney proximal tubule and the small intestine. It carries various di- and tri- carboxylates such as suc c i n a t e , - k e t o g l u t a r a t e a n d c i t r a t e . N a D C 1 i s i n v o l v e d i n r e g u l a t i n g t h e c o n c e n t r a t i o n o f t r i c a r b o x y l i c a c i d c y c l e i n t e r m e d i a t e s i n k i d n e y c e l l s a n d u r i n e . T h e r e f o r e , N a D C 1 i n f l u e n c e s t h e h o m e o s t a s i s o f c i t r a t e , w h i c h m a y b e a s s o c i a t e d w i t h t h e f o r m a t i o n of kidney stones. The studies in this dissertation focus on understanding various structural and functional aspects of NaDC1.
Previous studies indicate importance of TM 7, 10 and 11 for substrate binding. In this study, conserved prolines from TM 7 and 10 were mutated to alanine and glycine to understand structural as well as functional importance of these residues. Alanine is a strong (-helix former and is less flexible whereas glycine, with no side chain, is a strong helix breaker and is more flexible. If prolines in NaDC1 are responsible for kink formation and if kink is necessary for maintaining the stability of the transporter then mutating proline to glycine will have less adverse effect compared with mutating to alanine. This study indicates that Proline 327 in TM 7 when mutated to glycine was not able to reach the plasma membrane, showed no expression as well as succinate transport activity. Proline 351 plays an important role in cell surface regulation and protein trafficking. The prolines found in TM 10 at positions 523 and 524 do not appear to have functional roles but might be important for protein stability.
The current model of NaDC1, based on hydropathy analysis, contains 11 TM but secondary structure prediction algorithms predict at least 13 TM. To differentiate between the 11 and the 13 TM models, individual cysteine residues were substituted for other amino acids in predicted extracellular and intracellular loops of rbNaDC1. The extracellular accessibility of the cysteines was determined by chemical labeling with MTSEA-biotin [N-biotinylaminoethyl methanethiosulfonate]. Based on the site-directed chemical labeling experiments and computational homology modeling a modified model of NaDC1 was constructed containing 11 TM. Further studies indicate that mutants A39C, K84C, A133C, T252C, G356C, T482C and M493C were accessible in sodium buffer but their reactivity with methanethiosulfonate and their accessibility from outside changes in different buffer conditions in presence and absence of sodium and succinate. This indicates conformational changes in the transporter during the transport cycle. This new model will provide a structural framework towards understanding the structure-function relationship of NaDC1.
To identify conformationally sensitive residues in the Na+/dicarboxylate cotransporter that are accessible from both sides of the membrane, a bacterial homolog of NaDC1, Na+/dicarboxylate symporter (SdcS) from Staphylococcus aureus was used. The eukaryotic transporter could not be used due to experimental difficulties in studying accessibility of the transporter from inside the cell. Previous studies indicate that rbNaDC1 contains structurally, functionally and conformationally important residues such as Lys-84, Asp-373, Met-493. When protein sequence of rbNaDC1 was aligned with SdcS these residues correspond to Asn-108, Asp-329 and Leu-436 of SdcS. Cysteines were substituted at these locations in cysteineless SdcS and their accessibility was tested by MTSET reagent in right-side-out vesicles (RSO) and inside-out vesicles (ISO) in different conformational conditions such as in presence and absence of Na+ and substrate. SdcS showed similar succinate affinity in ISO and RSO vesicles but the transport system was asymmetric with Vmax of SdcS RSO is four times higher than Vmax of ISO. Residue Asn-108 was accessible from outside and inside in presence of Na+ only. The N108C mutant was not accessible with the conformational state in absence of Na+ and showed substrate protection likely due to steric hindrance by chemical labeling. Therefore, residue Asn-108 is probably located in a transmembrane helix near a water-filled pore or in a re-entrant loop accessible from both sides of the membrane.
In conclusion, this work provides a new insight into structural and functional aspects of Na+/dicarboxylate cotransporters. As limited high-resolution structural information is available about membrane transporters these biochemical and computational studies elucidate fundamental information of structural details of Na+/dicarboxylate cotransporters as well as help us in understanding its role in cellular functions.
TABLE OF CONTENTS
TOC \h \z \t "Heading 2,1,Heading 3,2,Heading 4,3" HYPERLINK \l "_Toc202693492" LIST OF TABLES PAGEREF _Toc202693492 \h xiii
HYPERLINK \l "_Toc202693493" LIST OF FIGURES PAGEREF _Toc202693493 \h xiv
HYPERLINK \l "_Toc202693496" LIST OF ABBREVIATIONS PAGEREF _Toc202693496 \h xviii
HYPERLINK \l "_Toc202693497" CHAPTER 1: INTRODUCTION PAGEREF _Toc202693497 \h 1
HYPERLINK \l "_Toc202693498" MEMBRANE TRANSPORT PROTEINS PAGEREF _Toc202693498 \h 1
HYPERLINK \l "_Toc202693499" STRUCTURAL BIOLOGY OF MEMBRANE TRANSPORTERS PAGEREF _Toc202693499 \h 4
HYPERLINK \l "_Toc202693500" TRANSLOCATION MECHANISM BY MEMBRANE TRANSPORTERS PAGEREF _Toc202693500 \h 6
HYPERLINK \l "_Toc202693501" THE SOLUTE CARRIER 13 GENE FAMILY PAGEREF _Toc202693501 \h 8
HYPERLINK \l "_Toc202693502" THE LOW AFFINITY Na+/DICARBOXYLATE COTRANSPORTER, NaDC1 PAGEREF _Toc202693502 \h 10
HYPERLINK \l "_Toc202693503" Isolation of NaDC1 cDNA PAGEREF _Toc202693503 \h 10
HYPERLINK \l "_Toc202693504" Tissue distribution of NaDC1 PAGEREF _Toc202693504 \h 10
HYPERLINK \l "_Toc202693505" Substrate specificity PAGEREF _Toc202693505 \h 11
HYPERLINK \l "_Toc202693506" Cation sensitivity PAGEREF _Toc202693506 \h 11
HYPERLINK \l "_Toc202693507" Functional characterization of NaDC1 PAGEREF _Toc202693507 \h 12
HYPERLINK \l "_Toc202693508" Na+-coupled transport mechanism of NaDC1 PAGEREF _Toc202693508 \h 12
HYPERLINK \l "_Toc202693509" Secondary structure model of NaDC1 PAGEREF _Toc202693509 \h 14
HYPERLINK \l "_Toc202693510" Structure-function studies of NaDC1 PAGEREF _Toc202693510 \h 15
HYPERLINK \l "_Toc202693511" Physiological significance of NaDC1 PAGEREF _Toc202693511 \h 18
HYPERLINK \l "_Toc202693512" THE Na+/DICARBOXYLATE SYMPORTER FROM Staphylococcus aureus PAGEREF _Toc202693512 \h 21
HYPERLINK \l "_Toc202693513" AIMS OF THE STUDIES IN THIS DISSERTATION PAGEREF _Toc202693513 \h 22
HYPERLINK \l "_Toc202693514" Aim 1: To determine the role of conserved proline residues in the structure and function of Na+/dicarboxylate cotransporter 1 PAGEREF _Toc202693514 \h 22
HYPERLINK \l "_Toc202693515" Aim 2: To determine the topology of Na+/dicarboxylate cotransporter 1 using site-directed chemical labeling. PAGEREF _Toc202693515 \h 23
HYPERLINK \l "_Toc202693516" Aim 3: Identification of conformationally sensitive amino acids in the Na+/dicarboxylate symporter. PAGEREF _Toc202693516 \h 23
HYPERLINK \l "_Toc202693517" Aim 4: Identification of determinants of citrate binding and transport in the bacterial Na+/dicarboxylate symporter using random mutagenesis approach. PAGEREF _Toc202693517 \h 24
HYPERLINK \l "_Toc202693518" CHAPTER 2: ROLE OF CONSERVED PROLINES IN THE STRUCTURE AND FUNCTION OF THE Na+/DICARBOXYLATE COTRANSPORTER 1 PAGEREF _Toc202693518 \h 25
HYPERLINK \l "_Toc202693519" INTRODUCTION PAGEREF _Toc202693519 \h 25
HYPERLINK \l "_Toc202693520" MATERIALS AND METHODS PAGEREF _Toc202693520 \h 26
HYPERLINK \l "_Toc202693521" Site-directed mutagenesis PAGEREF _Toc202693521 \h 26
HYPERLINK \l "_Toc202693522" Expression of rbNaDC1 Mutants in HRPE Cells PAGEREF _Toc202693522 \h 27
HYPERLINK \l "_Toc202693523" Transport Assays PAGEREF _Toc202693523 \h 28
HYPERLINK \l "_Toc202693524" Dual-label Competitive Transport Experiments PAGEREF _Toc202693524 \h 28
HYPERLINK \l "_Toc202693525" Cell Surface Biotinylation and Total Protein Expression PAGEREF _Toc202693525 \h 29
HYPERLINK \l "_Toc202693526" RESULTS PAGEREF _Toc202693526 \h 32
HYPERLINK \l "_Toc202693527" Proline to Alanine and Glycine Mutations in rbNaDC1 PAGEREF _Toc202693527 \h 32
HYPERLINK \l "_Toc202693528" Protein Expression and Transport Activity of Proline Mutants PAGEREF _Toc202693528 \h 32
HYPERLINK \l "_Toc202693529" Effect of Chemical Chaperones on Inactive TM 7 Mutants PAGEREF _Toc202693529 \h 34
HYPERLINK \l "_Toc202693530" Functional Characteristics of Proline Mutants PAGEREF _Toc202693530 \h 34
HYPERLINK \l "_Toc202693532" DISCUSSION PAGEREF _Toc202693532 \h 44
HYPERLINK \l "_Toc202693533" CHAPTER 3: A MEMBRANE TOPOLOGY MODEL OF Na+/ DICARBOXYLATE COTRANSPORTER 1 PAGEREF _Toc202693533 \h 49
HYPERLINK \l "_Toc202693534" INTRODUCTION PAGEREF _Toc202693534 \h 49
HYPERLINK \l "_Toc202693535" MATERIALS AND METHODS PAGEREF _Toc202693535 \h 50
HYPERLINK \l "_Toc202693536" Plasmid Constructs and Site-directed Mutagenesis PAGEREF _Toc202693536 \h 50
HYPERLINK \l "_Toc202693537" Expression of Cysteine Mutants in HRPE Cells PAGEREF _Toc202693537 \h 51
HYPERLINK \l "_Toc202693538" Transport Assays PAGEREF _Toc202693538 \h 52
HYPERLINK \l "_Toc202693539" Chemical Labeling with MTSET PAGEREF _Toc202693539 \h 52
HYPERLINK \l "_Toc202693540" Cell Surface Biotinylations PAGEREF _Toc202693540 \h 52
HYPERLINK \l "_Toc202693541" Topology Assay: Labeling of Cysteine Mutants with MTSEA-biotin PAGEREF _Toc202693541 \h 53
HYPERLINK \l "_Toc202693542" Topology Prediction and Homology Modeling PAGEREF _Toc202693542 \h 54
HYPERLINK \l "_Toc202693543" RESULTS PAGEREF _Toc202693543 \h 55
HYPERLINK \l "_Toc202693544" The 11 versus 13 TM Topology Models for NaDC1 PAGEREF _Toc202693544 \h 55
HYPERLINK \l "_Toc202693545" Protein Expression and Transport Activity of Cysteine Mutants: C476S background PAGEREF _Toc202693545 \h 56
HYPERLINK \l "_Toc202693546" Sensitivity to MTSET: C476S background PAGEREF _Toc202693546 \h 57
HYPERLINK \l "_Toc202693547" Cysteine Accessibility to Methanethiosulfonate Reagents: C476S background PAGEREF _Toc202693547 \h 57
HYPERLINK \l "_Toc202693548" Protein Expression and Transport Activity of Cysteine Mutants: 4N background PAGEREF _Toc202693548 \h 59
HYPERLINK \l "_Toc202693549" Sensitivity to MTSET: 4N background PAGEREF _Toc202693549 \h 59
HYPERLINK \l "_Toc202693550" MTSET Sensitivity of 4N Cysteine Mutants with Different Cations and Substrate PAGEREF _Toc202693550 \h 60
HYPERLINK \l "_Toc202693551" Cysteine Labeling by MTSEA-biotin: 4N background PAGEREF _Toc202693551 \h 61
HYPERLINK \l "_Toc202693552" Effect of Cations and Substrate on Labeling of Cysteine-Substituted Mutants by MTSEA-biotin PAGEREF _Toc202693552 \h 62
HYPERLINK \l "_Toc202693554" DISCUSSION PAGEREF _Toc202693554 \h 75
HYPERLINK \l "_Toc202693555" CHAPTER 4: IDENTIFICATION OF CONFORMATIONALLY SENSITIVE AMINO ACIDS IN THE Na+/DICARBOXYLATE SYMPORTER PAGEREF _Toc202693555 \h 83
HYPERLINK \l "_Toc202693556" INTRODUCTION PAGEREF _Toc202693556 \h 83
HYPERLINK \l "_Toc202693557" MATERIALS AND METHODS PAGEREF _Toc202693557 \h 84
HYPERLINK \l "_Toc202693558" Site-directed Mutagenesis PAGEREF _Toc202693558 \h 84
HYPERLINK \l "_Toc202693559" Preparation of Membrane Vesicles PAGEREF _Toc202693559 \h 85
HYPERLINK \l "_Toc202693560" Transport Assays PAGEREF _Toc202693560 \h 87
HYPERLINK \l "_Toc202693561" Immunoblot Analysis PAGEREF _Toc202693561 \h 88
HYPERLINK \l "_Toc202693562" Protein Determination PAGEREF _Toc202693562 \h 89
HYPERLINK \l "_Toc202693563" RESULTS PAGEREF _Toc202693563 \h 89
HYPERLINK \l "_Toc202693564" Assay of Na+ Dependent Succinate Transport PAGEREF _Toc202693564 \h 89
HYPERLINK \l "_Toc202693565" Vesicle Orientation PAGEREF _Toc202693565 \h 90
HYPERLINK \l "_Toc202693566" Na+ Activation of Succinate Uptakes in RSO and ISO Membrane Vesicles PAGEREF _Toc202693566 \h 90
HYPERLINK \l "_Toc202693567" Kinetics of Succinate Transport PAGEREF _Toc202693567 \h 91
HYPERLINK \l "_Toc202693568" Mutagenesis of SdcS PAGEREF _Toc202693568 \h 91
HYPERLINK \l "_Toc202693569" Protein Expression and Transport Activity of Cysteine-Substituted Mutants N108C, D329C and L436C PAGEREF _Toc202693569 \h 92
HYPERLINK \l "_Toc202693570" Succinate Kinetics in Cysteine-Substituted Mutants PAGEREF _Toc202693570 \h 93
HYPERLINK \l "_Toc202693571" MTSET Sensitivity of Cysteine-Substituted Mutants PAGEREF _Toc202693571 \h 94
HYPERLINK \l "_Toc202693572" Effect of Temperature on MTSET inhibition PAGEREF _Toc202693572 \h 95
HYPERLINK \l "_Toc202693577" DISCUSSION PAGEREF _Toc202693577 \h 104
HYPERLINK \l "_Toc202693578" CHAPTER 5: IDENTIFICATION OF DETERMINANTS OF CITRATE BINDING AND TRANSPORT IN THE BACTERIAL Na+/DICARBOXYLATE SYMPORTER USING A RANDOM MUTAGENESIS APPROACH PAGEREF _Toc202693578 \h 111
HYPERLINK \l "_Toc202693579" INTRODUCTION PAGEREF _Toc202693579 \h 111
HYPERLINK \l "_Toc202693580" MATERIALS AND METHODS PAGEREF _Toc202693580 \h 113
HYPERLINK \l "_Toc202693581" Site-directed Mutagenesis PAGEREF _Toc202693581 \h 113
HYPERLINK \l "_Toc202693582" Random Mutagenesis and Screening of Citrate Transporter PAGEREF _Toc202693582 \h 113
HYPERLINK \l "_Toc202693583" Whole cell Transport Assays PAGEREF _Toc202693583 \h 115
HYPERLINK \l "_Toc202693584" Preparation of right-side-out vesicles and transport assays PAGEREF _Toc202693584 \h 115
HYPERLINK \l "_Toc202693585" RESULTS PAGEREF _Toc202693585 \h 117
HYPERLINK \l "_Toc202693586" Random Mutagenesis of SdcS to Identify Determinants of Citrate Binding PAGEREF _Toc202693586 \h 117
HYPERLINK \l "_Toc202693587" Random Mutagenesis of SdcS Mutants, SdcS-MASN and SdcS-LASS to Identify Determinants of Citrate binding PAGEREF _Toc202693587 \h 119
HYPERLINK \l "_Toc202693588" R a n d o m M u t a g e n e s i s o f S d c S t o i d e n t i f y D e t e r m i n a n t s o f C i t r a t e a n d - K e t o g l u t a r a t e B i n d i n g P A G E R E F _ T o c 2 0 2 6 9 3 5 8 8 \ h 1 2 0
H Y P E R L I N K \ l " _ T o c 2 0 2 6 9 3 5 8 9 " D I S C U S S I O N P A G E R E F _ T o c 2 0 2 6 9 3 5 8 9 \ h 1 2 8
H Y P E R L I N K \ l " _ T o c 2 0 2 6 9 3 5 9 0 " C H A P T E R 6 : C O N C L U SIONS AND FUTURE DIRECTIONS PAGEREF _Toc202693590 \h 132
HYPERLINK \l "_Toc202693591" REFERENCES PAGEREF _Toc202693591 \h 140
HYPERLINK \l "_Toc202693592" VITA PAGEREF _Toc202693592 \h 156
HIDDEN TEXT: If you choose to place the chapter number (Chapter 1) and the chapter title (Introduction) on different lines, the automatically generated table of contents will reflect that format. After creating a new table of contents, set them on the same line by deleting the page number and paragraph marker at the end of each chapter number line.
LIST OF TABLES
TOC \h \z \t "Caption + 12 pt" \c HYPERLINK \l "_Toc199320474" Table 1.1 General characteristics of members of the SLC13 family PAGEREF _Toc199320474 \h 9
HYPERLINK \l "_Toc199320475" Table 2.1 Succinate kinetics of rbNaDC1 and double mutant P351A-F347P PAGEREF _Toc199320475 \h 36
HYPERLINK \l "_Toc199320476" Table 4.1 Succinate kinetics of wild-type SdcS, cysteineless C457S and cysteine-substituted mutants, N108C, D329C, L436C in right-side-out (RSO) and inside-out (ISO) membrane vesicles from E. coli PAGEREF _Toc199320476 \h 96
LIST OF FIGURES
TOC \h \z \t "Caption + 11 pt" \c HYPERLINK \l "_Toc199319988" Figure 1.1 General classification of membrane transport proteins. PAGEREF _Toc199319988 \h 2
HYPERLINK \l "_Toc199319989" Figure 1.2 Transport model of Na+/dicarboxylate cotransporter. PAGEREF _Toc199319989 \h 13
HYPERLINK \l "_Toc199319990" Figure 1.3 Secondary structure model of NaDC1... PAGEREF _Toc199319990 \h 15
HYPERLINK \l "_Toc199319991" Figure 1.4 Schematic representation of dicarboxylate transport in renal proximal tubules. PAGEREF _Toc199319991 \h 21
HYPERLINK \l "_Toc199319992" Figure 2.1 Multiple sequence alignments of transmembrane helices 7 and 10 from rbNaDC1 with some members of SLC13 gene family. PAGEREF _Toc199319992 \h 31
HYPERLINK \l "_Toc199319993" Figure 2.2 Western blots of (A) cell surface and (B) total biotinylated protein expression. PAGEREF _Toc199319993 \h 37
HYPERLINK \l "_Toc199319994" Figure 2.3 Activity and expression of single (A) and double (B) mutants of rbNaDC1. PAGEREF _Toc199319994 \h 38
HYPERLINK \l "_Toc199319996" Figure 2.4 Treatment of non-expressing mutants with glycerol.. PAGEREF _Toc199319996 \h 39
HYPERLINK \l "_Toc199319997" Figure 2.5 Effect of glycerol on TM 7 mutants. PAGEREF _Toc199319997 \h 40
HYPERLINK \l "_Toc199319998" Figure 2.6 Time course of competitive uptake of succinate and citrate in wild type rbNaDC1 and Transport specificity ratios. PAGEREF _Toc199319998 \h 41
HYPERLINK \l "_Toc199319999" Figure 2.7 Transport specificity ratio (TSR) of proline mutants compared with wild type rbNaDC1.. PAGEREF _Toc199319999 \h 42
HYPERLINK \l "_Toc199320000" Figure 2.8 Transport specificity ratio (TSR) of mutants treated with glycerol to improve expression. PAGEREF _Toc199320000 \h 43
HYPERLINK \l "_Toc199320001" Figure 2.9 Secondary structure model of NaDC1 showing the location of prolines in TM 7 and 10 that were mutated in this study. PAGEREF _Toc199320001 \h 48
HYPERLINK \l "_Toc199320002" Figure 3.1 Comparison of the 11 TM versus 13 TM topology models for rbNaDC1. PAGEREF _Toc199320002 \h 64
HYPERLINK \l "_Toc199320003" Figure 3.2 Western blots of cell surface protein expression of cysteine mutants substituted in the NaDC1/C476S parental transporter and Succinate transport activity and protein expression of cysteine-substituted mutants made in C476S background expressed in HRPE cells. PAGEREF _Toc199320003 \h 65
HYPERLINK \l "_Toc199320004" Figure 3.3 Effect of 1 mM MTSET on succinate transport by cysteine-substituted mutants: C476S background. PAGEREF _Toc199320004 \h 66
HYPERLINK \l "_Toc199320005" Figure 3.4 Labeling of cysteine-substituted mutants (C476S background)with MTSEA-biotin. PAGEREF _Toc199320005 \h 67
HYPERLINK \l "_Toc199320006" Figure 3.5 MTSEA-biotin labeling of G367C mutant made in C476S and 4N parental transporters. PAGEREF _Toc199320006 \h 68
HYPERLINK \l "_Toc199320007" Figure 3.6 Western blots of cell surface protein expression of cysteine mutants substituted in the NaDC1/4N parental transporter. Transport activity and cell surface protein abundance of cysteine-substituted mutants in the 4N parental transporter. PAGEREF _Toc199320007 \h 69
HYPERLINK \l "_Toc199320008" Figure 3.7 Effect of 1 mM MTSET on succinate transport by cysteine-substituted mutants: 4N background. PAGEREF _Toc199320008 \h 70
HYPERLINK \l "_Toc199320009" Figure 3.8 Effect of substrate and cation on sensitivity of cysteine-substituted mutants to MTSET labeling: 4N background. PAGEREF _Toc199320009 \h 71
HYPERLINK \l "_Toc199320010" Figure 3.9 Labeling of substituted cysteine residues (4N background) with MTSEA-biotin. PAGEREF _Toc199320010 \h 72
HYPERLINK \l "_Toc199320011" Figure 3.10 Labeling of substituted cysteine mutants ( G 4 2 9 C / 4 N , G 4 3 1 C / 4 N , A 4 3 3 C / 4 N , G 4 3 7 C / 4 N a n d M 4 9 3 C / 4 N ) w i t h E Z - L i n k "! P E O - M a l e i m i d e A c t i v a t e d B i o t i n . P A G E R E F _ T o c 1 9 9 3 2 0 0 1 1 \ h 7 3
H Y P E R L I N K \ l " _ T o c 1 9 9 3 2 0 0 1 2 " F i g u r e 3 . 1 1 M T S E A - b i o t i n l a b e l i n g o f c y s t e i n e - s u b s t i t u t e d 4 N m u t a n t s u n d e r d i f f e r e n t b u ffer conditions. PAGEREF _Toc199320012 \h 74
HYPERLINK \l "_Toc199320013" Figure 3.12 Revised topology model of rbNaDC1. PAGEREF _Toc199320013 \h 81
HYPERLINK \l "_Toc199320014" Figure 3.13 Model of three dimensional structure of rbNaDC1 containing 11 TM. PAGEREF _Toc199320014 \h 82
HYPERLINK \l "_Toc199320015" Figure 4.1 Time course of succinate uptake by SdcS right-side-out and inside-out membrane vesicles. PAGEREF _Toc199320015 \h 97
HYPERLINK \l "_Toc199320016" Figure 4.2 Effect of methanethiosulfonate reagents on succinate uptake by SdcS in right-side-out (RSO) and inside-out (ISO) membrane vesicles. PAGEREF _Toc199320016 \h 98
HYPERLINK \l "_Toc199320017" Figure 4.3 Na+-activation of succinate uptake. PAGEREF _Toc199320017 \h 99
HYPERLINK \l "_Toc199320018" Figure 4.4 Sequence alignment of rabbit NaDC1 with SdcS. PAGEREF _Toc199320018 \h 100
HYPERLINK \l "_Toc199320019" Figure 4.5 Western blot of right-side-out (RSO) and inside-out membrane vesicles (ISO) expressing pQE-80L, SdcS, cysteineless C457S, D329C, L436C and N108C.. PAGEREF _Toc199320019 \h 100
HYPERLINK \l "_Toc199320020" Figure 4.6 Activity and expression of right-side-out and inside-out membrane vesicles: SdcS, cysteineless C457S, D329C, L436C and N108C PAGEREF _Toc199320020 \h 101
HYPERLINK \l "_Toc199320021" Figure 4.7 Effect of MTSET on succinate transport by cysteine-substituted SdcS mutants in right-side-out and inside-out membrane vesicles. PAGEREF _Toc199320021 \h 102
HYPERLINK \l "_Toc199320022" Figure 4.8 Temperature dependence of MTSET labeling on N108C right-side-out mutant PAGEREF _Toc199320022 \h 103
HYPERLINK \l "_Toc199320023" Figure 4.9 Secondary structure model of SdcS. PAGEREF _Toc199320023 \h 110
HYPERLINK \l "_Toc199320024" Figure 5.1 The random mutagenesis approach using the GeneMorph II EZClone kit PAGEREF _Toc199320024 \h 116
HYPERLINK \l "_Toc199320025" Figure 5.2 Citrate transport activity of S d c S r a n d o m m u t a n t s t r a n s f o r m e d i n t o X L 1 0 - G o l d s t r a i n o f E . c o l i . P A G E R E F _ T o c 1 9 9 3 2 0 0 2 5 \ h 1 2 2
H Y P E R L I N K \ l " _ T o c 1 9 9 3 2 0 0 2 6 " F i g u r e 5 . 3 T r a n s p o r t a c t i v i t y o f S d c S r a n d o m m u t a n t s t r a n s f o r m e d i n t o D H 5 s t r a i n o f E . c o l i . P A G E R E F _ T o c 1 9 9 3 2 0 0 2 6 \ h 123
HYPERLINK \l "_Toc199320027" Figure 5.4 Sequence alignment of rabbit NaDC1 with SdcS. PAGEREF _Toc199320027 \h 124
HYPERLINK \l "_Toc199320028" Figure 5.5 Transport activity of SdcS random mutants.. PAGEREF _Toc199320028 \h 124
HYPERLINK \l "_Toc199320029" Figure 5.6 Transport activity of SdcS and mutants (numbered 13-34) in right-side-out membrane vesicles of E. coli (BL21 strain). PAGEREF _Toc199320029 \h 125
HYPERLINK \l "_Toc199320030" Figure 5.7 Time course of su c c i n a t e a n d - k e t o g l u t a r a t e u p t a k e b y r i g h t - s i d e - o u t m e m b r a n e v e s i c l e s o f E . c o l i ( B L 2 1 s t r a i n ) h o u s i n g p Q E - 8 0 L , S d c S , S d c S r a n d o m m u t a n t s 1 7 a n d 1 9 . P A G E R E F _ T o c 1 9 9 3 2 0 0 3 0 \ h 1 2 6
H Y P E R L I N K \ l " _ T o c 1 9 9 3 2 0 0 3 1 " F i g u r e 5 . 8 T i m e c o u r s e o f - k e t o g l u t a rate uptake by right-side-out membrane vesicles of E. coli (BL21 strain) housing SdcS, SdcS random mutants 17 and 19. Na+ verses choline uptake. PAGEREF _Toc199320031 \h 127
LIST OF ABBREVIATIONS
cDNA complementary deoxyribonucleic acid
2, 2-DMS 2, 2-dimethylsuccinate
f Prefix, flounder
h Prefix, human
HEPES N-2-hydroxyethylpiperazine-n-2'-ethanesulfonic acid
Indy Na+-independent dicarboxylate transporter from Drosophila
IgG Immunoglobin G
kDa kilodalton
kb kilobase
m Prefix, mouse
MTSEA 2-aminoethylmethane thiosulfonate
MTSES 2-sulfonatoethylmethane thiosulfonate
MTSET 2-trimethylammonieothylmethane thiosulfonate
NaDC1 Low-affinity Na+/dicarboxylate cotransporter
NaDC3 High-affinity Na+/dicarboxylate cotransporter
NaCT Na+-coupled citrate transporter
NaS Na+/sulfate cotransporter
o Prefix, opossum
OAT Organic anion transporter
PBS Phosphate-buffered saline
PCR Polymerase chain reaction
LIST OF ABBREVIATIONS (CONT.)
PMSF Phenylmethanesulfonyl fluoride
r Prefix, ratLIST OF ABBREVIATION LIST OF ABBREVIATION LIST OF ABBREVIATION LIST OF ABBREVIATION LIST OF ABBREVIATION
rb Prefix, rabbit
RT-PCR Reverse transcription-polymerase chain reaction
SLC Solute carrier gene family
SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis
Sulfo-NHS-LC-Biotin Sulfosuccinimidyl-6-(biotinamido) hexanoate
TM Transmembrane helix
TSR Transport specificity ratio
x Prefix, Xenopus laevis
CHAPTER 1: INTRODUCTION
This dissertation focuses on understanding structural and functional aspects of the Na+/dicarboxylate cotransporters, NaDC. The first aim of this dissertation (Chapter 2) was to understand the role of conserved proline residues in Na+/dicarboxylate cotransporter 1 (NaDC1). In the second aim (Chapter 3), the topology of NaDC1 was determined by site-directed chemical labeling. The third aim (Chapter 4) was to identify and characterize conformationally sensitive amino acids in a homolog of NaDC1 from Staphylococcus aureus, Na+/dicarboxylate symporter (SdcS). In the final aim (Chapter 5), an attempt was made to identify determinants of citrate binding and transport by using random mutagenesis in SdcS. This introduction begins with general information about membrane transport systems, high-resolution structures of various membrane transporters available, mechanisms of substrate transport, the solute carrier 13 (SLC 13) gene family members and finally NaDC1 as well as SdcS transporters.
MEMBRANE TRANSPORT PROTEINS
It was only in the 1950s that the first reports began to appear in the literature indicating that the movement of a number of substances, both uncharged and ionic, across cell membranes was catalyzed by specific proteins. The kinetics (and selectivity) of some of these proteins were worked out in great detail, particularly for amino acids in Ehrlich ascites cells ADDIN REFMGR.CITE Widdas195269Inability of diffusion to account for placental glucose transfer in the sheep and consideration of the kinetics of a possible carrier transferJournal69Inability of diffusion to account for placental glucose transfer in the sheep and consideration of the kinetics of a possible carrier transferWiddas,W.F.1952/9GlucosephysiologyPlacentaNot in File2339J.Physiol1181PM:13000688J.Physiol1(1) and for monosaccharides in human erythrocytes ADDIN REFMGR.CITE LeFevre1961821Sugar transport in the red blood cell: structure-activity relationships in substrates and antagonistsJournal821Sugar transport in the red blood cell: structure-activity relationships in substrates and antagonistsLeFevre,P.G.1961/3bloodBlood GlucoseErythrocytesGlucosemetabolismtransportNot in File3970Pharmacol.Rev.13PM:13760340Pharmacol.Rev.1(2). Later due to increase in the membrane transport protein gene sequences and structures of the proteins, in 1990s the first draft of a classification system was put forward. Further in 1999 and updated in 2002, a novel classification system of membrane transport proteins was proposed by Milton H. Saier Jr. known as Transport Classification (TC) system ADDIN REFMGR.CITE Busch2002822The transporter classification (TC) system, 2002Journal822The transporter classification (TC) system, 2002Busch,W.Saier,M.H.,Jr.2002AnimalsBacterial ProteinsBiological TransportclassificationEvolution,MolecularHumansmetabolismProtein TransportProteinstransportNot in File287337Crit Rev.Biochem.Mol.Biol.375Division of Biology, University of California at San Diego, La Jolla 92093-0116, USAPM:12449427Crit Rev.Biochem.Mol.Biol.1Busch20041The IUBMB-endorsed transporter classification systemJournal1The IUBMB-endorsed transporter classification systemBusch,W.Saier,M.H.,Jr.2004/7Amino Acid SequenceBiochemistrychemistryclassificationDatabases,ProteinDocumentationEvolution,MolecularGuidelines as TopicInternationalityMembrane Transport ProteinsmethodsMolecular BiologyMolecular Sequence DataProteinsstandardsTerminology as TopictransportVocabulary,ControlledNot in File253262Mol.Biotechnol.273Division of Biological Sciences, University of California at San Diego, La Jolla, CA 92093-0116, USAPM:15247498Mol.Biotechnol.1Saier1999590Genome archeology leading to the characterization and classification of transport proteinsJournal590Genome archeology leading to the characterization and classification of transport proteinsSaier,M.H.1999/10Carrier ProteinsclassificationgeneticsGenome,ArchaealGenome,BacterialmetabolismPhylogenyProteinsSubstrate SpecificitySupport,Non-U.S.Gov'tSupport,U.S.Gov't,P.H.S.transportNot in File555561Curr.Opin.Microbiol.25Department of Biology University of California at San Diego La Jolla, CA 92093-0116, USA. msaier@ucsd.eduPM:10508720Curr.Opin.Microbiol.1Saier2000591A functional-phylogenetic classification system for transmembrane solute transportersJournal591A functional-phylogenetic classification system for transmembrane solute transportersSaier,M.H.,Jr.2000classificationNot in File354411Microbiology and Molecular Biology Reviews64No.2Microbiology and Molecular Biology Reviews1Saier2000685Families of transmembrane sugar transport proteinsJournal685Families of transmembrane sugar transport proteinsSaier,M.H.,Jr.2000/2ATP-Binding Cassette TransportersBacterial ProteinsBiological TransportCarbohydratesCarrier ProteinsMembrane ProteinsmetabolismMonosaccharide Transport ProteinsProteinsreviewsugar transportSupport,Non-U.S.Gov'tSupport,U.S.Gov't,P.H.S.transportNot in File699710Mol.Microbiol.354Department of Biology, University of California at San Diego, La Jolla, CA 92093-0116, USA. msaier@ucsd.eduPM:10692148Mol.Microbiol.1(3-7) which is based on functional and phylogenic characteristics of transport proteins. This classification system was formally adopted by the International Union of Biochemistry and Molecular Biology (IUBMB) in 2002. According to the TC system, the membrane transport proteins are classified into two major groups, namely channels and carriers (Figure 1.1).
Figure 1.1 General classification of membrane transport proteins. The transport proteins are classified according to TC system ADDIN REFMGR.CITE Busch2002822The transporter classification (TC) system, 2002Journal822The transporter classification (TC) system, 2002Busch,W.Saier,M.H.,Jr.2002AnimalsBacterial ProteinsBiological TransportclassificationEvolution,MolecularHumansmetabolismProtein TransportProteinstransportNot in File287337Crit Rev.Biochem.Mol.Biol.375Division of Biology, University of California at San Diego, La Jolla 92093-0116, USAPM:12449427Crit Rev.Biochem.Mol.Biol.1(3). Transport proteins are classified into two major types: channel proteins and carrier proteins. Carriers or transporters can be further sub-classified into three types: uniporters, primary active transporters and secondary active transporters.
Ion channels are selective hydrophilic pores that permit the movement of substrates across the membrane down their electrochemical gradients ADDIN REFMGR.CITE Hille198616Ionic channels: molecular pores of excitable membranesJournal16Ionic channels: molecular pores of excitable membranesHille,B.1986CalciumCell MembraneCell Membrane PermeabilityElectrophysiologyEvolutionIon ChannelsmetabolismphysiologyPotassiumReceptors,CholinergicReceptors,DrugSodiumNot in File4769Harvey Lect.82PM:2452140Harvey Lect.1(8). On the other hand, membrane transporters are classified into uniporters, primary active transporters and secondary active transporters based on the energy source for transport, nature of the transport and mode of transport ADDIN REFMGR.CITE Busch2002822The transporter classification (TC) system, 2002Journal822The transporter classification (TC) system, 2002Busch,W.Saier,M.H.,Jr.2002AnimalsBacterial ProteinsBiological TransportclassificationEvolution,MolecularHumansmetabolismProtein TransportProteinstransportNot in File287337Crit Rev.Biochem.Mol.Biol.375Division of Biology, University of California at San Diego, La Jolla 92093-0116, USAPM:12449427Crit Rev.Biochem.Mol.Biol.1(3). Uniporters such as glucose transporters (GLUTs) are facilitated diffusion carriers that move substrates down their electrochemical gradient. The primary active transporters (for example, the members of the ATP binding cassette family and P-type ATPases such as Na+/K+ ATPase) use energy from hydrolysis of adenosine triphosphate (ATP) directly to drive transport of solutes against their electrochemical gradient. The secondary active transporters carry solutes against their electrochemical gradient by coupling to electrochemical gradient generated by the primary active transport process. Major driving forces for secondary active transporters are Na+ and proton gradients. Depending on the direction of the solute transport secondary active transporters are classified into cotransporters (symporters) and exchangers (antiporters). In cotransporters, solutes are transported in same direction (e.g. NaDC1 uses the energy stored in the sodium gradient to transport three sodium ions and one dicarboxylate ion into the cell) whereas in exchangers solutes are transported in opposite directions (Na+/Ca+ exchanger uses the energy stored in the sodium gradient to transport two sodium ions into the cell and one calcium ion out of the cell). The major focus of this dissertation is on the NaDC transporters which belong to the secondary active cotransporter category.
STRUCTURAL BIOLOGY OF MEMBRANE TRANSPORTERS
Out of thousands of membrane proteins, high-resolution structures of only about 150 proteins are known, which contributes less than one percent of the total protein structures listed in the Protein Data Bank (PDB). There are several problems involved in crystallization of membrane proteins. Firstly, they are amphipathic in nature as they contain hydrophobic groups in contact with membrane phospholipids and hydrophilic groups in contact with the water. Therefore purifying membrane protein out of the lipid bilayer makes the protein nonfunctional and is challenging. Secondly, for crystallization studies overexpressed proteins are required and are difficult as very few mammalian membrane proteins have been functionally expressed in bacteria, the expression system used to acquire large quantities of proteins. Despite the difficulties in crystallization some high-resolution crystal structures are known.
The first three-dimensional structure of a secondary transporter, the E. coli AcrB transporter, was reported in 2002 ADDIN REFMGR.CITE Murakami200212Crystal structure of bacterial multidrug efflux transporter AcrBJournal12Crystal structure of bacterial multidrug efflux transporter AcrBMurakami,S.Nakashima,R.Yamashita,E.Yamaguchi,A.2002/10/10Bacterial Outer Membrane ProteinsBacterial ProteinsBiological TransportCarrier ProteinsCell MembranechemistryCrystallography,X-RayDrug Resistance,Multiple,BacterialEscherichia coliEscherichia coli ProteinsMembrane ProteinsMembrane Transport ProteinsmetabolismModels,MolecularMultidrug Resistance-Associated ProteinsProtein BindingProtein ConformationProtein Structure,TertiaryProteinsNot in File587593Nature4196907Department of Cell Membrane Biology, Institute of Scientific and Industrial Research, Osaka University, Ibaraki, Osaka 567-0047, JapanPM:12374972Nature1(9). AcrB is a resistance-nodulation cell division transporter (RND) that cooperates with a channel (TolC) to form a proton-driven multidrug exporter system. The structure of ligand bound AcrB was also solved and demonstrated that three ligand molecules can bind to the binding site near a central cavity ADDIN REFMGR.CITE Yu2003823AcrB multidrug efflux pump of Escherichia coli: composite substrate-binding cavity of exceptional flexibility generates its extremely wide substrate specificityJournal823AcrB multidrug efflux pump of Escherichia coli: composite substrate-binding cavity of exceptional flexibility generates its extremely wide substrate specificityYu,E.W.Aires,J.R.Nikaido,H.2003/10Anti-Bacterial AgentsBinding SitesCarrier Proteinsdrug effectsDrug Resistance,Multiple,BacterialEscherichia coliEscherichia coli ProteinsMembrane ProteinsmetabolismModels,MolecularMultidrug Resistance-Associated ProteinspharmacologyProteinsSubstrate SpecificityNot in File56575664J.Bacteriol.18519Department of Molecular and Cell Biology, University of California, Berkeley, California 94720-3202, USAPM:13129936Journal of BacteriologyJ.Bacteriol.1Yu2003824Structural basis of multiple drug-binding capacity of the AcrB multidrug efflux pumpJournal824Structural basis of multiple drug-binding capacity of the AcrB multidrug efflux pumpYu,E.W.McDermott,G.Zgurskaya,H.I.Nikaido,H.Koshland,D.E.,Jr.2003/5/9Anti-Infective AgentsAnti-Infective Agents,LocalBinding SitesCarrier ProteinsCell MembranechemistryChemistry,PhysicalCiprofloxacinCrystallizationCrystallography,X-RayDequaliniumElectrostaticsEscherichia coliEscherichia coli ProteinsEthidiumHydrogen BondingHydrophobicityisolation & purificationLigandsMembrane ProteinsmetabolismModels,MolecularMultidrug Resistance-Associated ProteinsProtein BindingProtein ConformationProtein Structure,QuaternaryProtein Structure,SecondaryProtein Structure,TertiaryProteinsRhodaminesNot in File976980Science3005621Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720-3202, USAPM:12738864Science1(10;11).
Later the three dimensional structures of E. coli lactose permease (LacY) and Glycerol-3-phosphate (GlpT) from the major facilitator superfamily were simultaneously reported in 2003 ADDIN REFMGR.CITE Abramson2003825Structure and mechanism of the lactose permease of Escherichia coliJournal825Structure and mechanism of the lactose permease of Escherichia coliAbramson,J.Smirnova,I.Kasho,V.Verner,G.Kaback,H.R.Iwata,S.2003/8/1Amino Acid SubstitutionBinding SitesBiological TransportCell MembranechemistryCrystallizationCrystallography,X-RayenzymologyEscherichia coliEscherichia coli ProteinsgeneticsHydrogen BondingHydrophobicityIon TransportLactoseMembrane Transport ProteinsmetabolismModels,MolecularMonosaccharide Transport ProteinsMutationProtein ConformationProtein Structure,SecondaryProtein Structure,TertiaryProteinsProtonsSubstrate SpecificitySymportersThiogalactosidestransportNot in File610615Science3015633Department of Biological Sciences, Imperial College London, London SW7 2AZ, UKPM:12893935Science1Huang2003681Structure and mechanism of the glycerol-3-phosphate transporter from Escherichia coliJournal681Structure and mechanism of the glycerol-3-phosphate transporter from Escherichia coliHuang,Y.Lemieux,M.J.Song,J.Auer,M.Wang,D.N.2003/8/1Amino Acid SequenceBinding SitesBiological TransportCell MembranechemistryCrystallizationCrystallography,X-RayenzymologyEscherichia coliEscherichia coli ProteinsGlycerophosphatesHelix-Turn-Helix MotifshumanMembrane Transport ProteinsmetabolismModels,MolecularMolecular Sequence DataPeriplasmPhosphatesProtein ConformationProtein FoldingProtein Structure,SecondaryProtein Structure,TertiaryProteinsSpectrum Analysis,MassSupport,Non-U.S.Gov'tSupport,U.S.Gov't,P.H.S.Not in File616620Science3015633Skirball Institute of Biomolecular Medicine and Department of Cell Biology, New York University School of Medicine, 540 First Avenue, New York, NY 10016, USAPM:12893936Science1(12;13). Major facilitator superfamily transporters are single-polypeptide secondary carriers capable of transporting small solutes in response to chemiosmotic ion gradients ADDIN REFMGR.CITE Saier1999826The major facilitator superfamilyJournal826The major facilitator superfamilySaier,M.H.,Jr.Beatty,J.T.Goffeau,A.Harley,K.T.Heijne,W.H.Huang,S.C.Jack,D.L.Jahn,P.S.Lew,K.Liu,J.Pao,S.S.Paulsen,I.T.Tseng,T.T.Virk,P.S.1999/11Amino Acid SequenceAnimalsCarrier ProteinsclassificationHumansMolecular Sequence DataProteinssequencetransportNot in File257279J.Mol.Microbiol.Biotechnol.12Department of Biology, University of California at San Diego, La Jolla 92093-0116, USA. saier@ucsd.eduPM:10943556J.Mol.Microbiol.Biotechnol.1(14). LacY is the well-known H+/lactose symporter whereas GlpT catalyses glycerol-3-P/Pi exchange. The transporters are monomers and contain two homologous domains, each containing six transmembrane helices (TM). Their structures show a large hydrophilic cavity between the two domains that is open to the cytoplasm and closed to the periplasm. The substrate binding site is in the middle of the helices. These structures support the alternating access model for transport in which two domains move relative to each other and open the substrate binding site alternately to two sides of the membrane, that is towards the cytoplasm and periplasm ADDIN REFMGR.CITE Abramson2003825Structure and mechanism of the lactose permease of Escherichia coliJournal825Structure and mechanism of the lactose permease of Escherichia coliAbramson,J.Smirnova,I.Kasho,V.Verner,G.Kaback,H.R.Iwata,S.2003/8/1Amino Acid SubstitutionBinding SitesBiological TransportCell MembranechemistryCrystallizationCrystallography,X-RayenzymologyEscherichia coliEscherichia coli ProteinsgeneticsHydrogen BondingHydrophobicityIon TransportLactoseMembrane Transport ProteinsmetabolismModels,MolecularMonosaccharide Transport ProteinsMutationProtein ConformationProtein Structure,SecondaryProtein Structure,TertiaryProteinsProtonsSubstrate SpecificitySymportersThiogalactosidestransportNot in File610615Science3015633Department of Biological Sciences, Imperial College London, London SW7 2AZ, UKPM:12893935Science1Lemieux2004682The structural basis of substrate translocation by the Escherichia coli glycerol-3-phosphate transporter: a member of the major facilitator superfamilyJournal682The structural basis of substrate translocation by the Escherichia coli glycerol-3-phosphate transporter: a member of the major facilitator superfamilyLemieux,M.J.Huang,Y.Wang,D.N.2004/8Escherichia coliNot in File405412Curr.Opin.Struct.Biol.144Skirball Institute of Biomolecular Medicine and Department of Cell Biology, New York University School of Medicine, 540 First Avenue, New York, New York 10016, USAPM:15313233Curr.Opin.Struct.Biol.1(12;15).
In 2004 a structure of E. coli ammonia transporter, AmtB, was solved ADDIN REFMGR.CITE Khademi2004827Mechanism of ammonia transport by Amt/MEP/Rh: structure of AmtB at 1.35 AJournal827Mechanism of ammonia transport by Amt/MEP/Rh: structure of AmtB at 1.35 AKhademi,S.O'Connell,J.,IIIRemis,J.Robles-Colmenares,Y.Miercke,L.J.Stroud,R.M.2004/9/10Amino Acid SequenceAmmoniaBinding SitesBiochemistryBiological TransportBiophysicsCation Transport ProteinsCell MembranechemistryCrystallizationCrystallography,X-RayEscherichia coliEscherichia coli ProteinsgeneticsHydrogen BondingHydrogen-Ion ConcentrationHydrophobicityLiposomesMembrane PotentialsmetabolismModels,MolecularMolecular Sequence DataProtein ConformationProtein FoldingProtein Structure,QuaternaryProtein Structure,SecondaryProteinsQuaternary Ammonium CompoundsRh-Hr Blood-Group SystemSequence AlignmenttransportWaterNot in File15871594Science3055690Department of Biochemistry and Biophysics, S412C Genentech Hall, University of California-San Francisco, 600 16th Street, San Francisco, CA 94143-2240, USAPM:15361618Science1(16). The structure is similar to that of major facilitator family proteins (LacY and GlpT) with two structurally homologous domains with five TM in each. The ammonium ion (NH4+) binds to the AmtB and passage of ammonia (NH3) takes place leaving proton (H+) behind. Later in 2004 the structure of the glutamate transporter (Gltph) homolog from Pyrococcus horikoshii was reported ADDIN REFMGR.CITE Yernool2004828Structure of a glutamate transporter homologue from Pyrococcus horikoshiiJournal828Structure of a glutamate transporter homologue from Pyrococcus horikoshiiYernool,D.Boudker,O.Jin,Y.Gouaux,E.2004/10/14Amino Acid SequenceAmino Acid Transport System X-AGBinding SitesBiochemistryBiophysicschemistryCrystallography,X-RaygeneticsGlutamic AcidmetabolismModels,MolecularMolecular Sequence DataMutationProtein Structure,QuaternaryProtein SubunitsPyrococcus horikoshiitransportNot in File811818Nature4317010Department of Biochemistry and Molecular Biophysics, Columbia University, 650 West 168th Street, New York, New York 10032, USAPM:15483603Nature1(17). In vertebrates, the glutamate transporter plays a major role in the uptake of glutamate from the synaptic cleft in the central nervous system. The glutamate transporter forms a trimeric complex with a bowl-shape structure ADDIN REFMGR.CITE Yernool2004828Structure of a glutamate transporter homologue from Pyrococcus horikoshiiJournal828Structure of a glutamate transporter homologue from Pyrococcus horikoshiiYernool,D.Boudker,O.Jin,Y.Gouaux,E.2004/10/14Amino Acid SequenceAmino Acid Transport System X-AGBinding SitesBiochemistryBiophysicschemistryCrystallography,X-RaygeneticsGlutamic AcidmetabolismModels,MolecularMolecular Sequence DataMutationProtein Structure,QuaternaryProtein SubunitsPyrococcus horikoshiitransportNot in File811818Nature4317010Department of Biochemistry and Molecular Biophysics, Columbia University, 650 West 168th Street, New York, New York 10032, USAPM:15483603Nature1(17). The actual substrate binding site is in between two reentrant loops. Translocation is carried out by opening and closing access to the substrate binding site by movement of the reentrant loops ADDIN REFMGR.CITE Brocke2002829Proximity of two oppositely oriented reentrant loops in the glutamate transporter GLT-1 identified by paired cysteine mutagenesisJournal829Proximity of two oppositely oriented reentrant loops in the glutamate transporter GLT-1 identified by paired cysteine mutagenesisBrocke,L.Bendahan,A.Grunewald,M.Kanner,B.I.2002/2/8Binding SitesBiochemistryCadmiumchemistryCysteineDithiothreitolExcitatory Amino Acid Transporter 2geneticsHela CellsHumansmetabolismMutagenesisProtein ConformationSodiumtransportNot in File39853992J.Biol.Chem.2776Department of Biochemistry, Hadassah Medical School, Hebrew University, Jerusalem 91120, IsraelPM:11724778The Journal of Biological ChemistryJ.Biol.Chem.JBC1Yernool2004828Structure of a glutamate transporter homologue from Pyrococcus horikoshiiJournal828Structure of a glutamate transporter homologue from Pyrococcus horikoshiiYernool,D.Boudker,O.Jin,Y.Gouaux,E.2004/10/14Amino Acid SequenceAmino Acid Transport System X-AGBinding SitesBiochemistryBiophysicschemistryCrystallography,X-RaygeneticsGlutamic AcidmetabolismModels,MolecularMolecular Sequence DataMutationProtein Structure,QuaternaryProtein SubunitsPyrococcus horikoshiitransportNot in File811818Nature4317010Department of Biochemistry and Molecular Biophysics, Columbia University, 650 West 168th Street, New York, New York 10032, USAPM:15483603Nature1(17;18).
In 2005, the crystal structure of the leucine transporter (LeuTAa) from Aquifex aeolicus was reported at 1.65 ADDIN REFMGR.CITE Yamashita2005819Crystal structure of a bacterial homologue of Na+/Cl- dependent neurotransmitter transportersJournal819Crystal structure of a bacterial homologue of Na+/Cl- dependent neurotransmitter transportersYamashita,A.Singh,S.K.Kawate,T.Jin,Y.Gouaux,E.2005/9/8Amino Acid SequenceBacteriaBacterial ProteinsBinding SitesBiological TransportBiophysicschemistryChloridesCrystallography,X-RayHydrophobicityLeucineMembrane Transport ProteinsmetabolismModels,MolecularMolecular Sequence DataNeurotransmitter AgentsProteinsSequence AlignmentSodiumStructure-Activity RelationshipWaterNot in File215223Nature4377056Department of Biochemistry and Molecular Biophysics andPM:16041361Nature1(19). This bacterial structure has made a major contribution in our knowledge of the structure of mammalian membrane proteins as structural details from LeuTAa have provided a framework to understand the molecular motion within the structure of serotonin transporter (SERT) ADDIN REFMGR.CITE Rudnick200636Serotonin transporters--structure and functionJournal36Serotonin transporters--structure and functionRudnick,G.2006Amino Acid SequenceAnimalsBacterial ProteinsBinding SiteschemistrygeneticsHumansIn VitroMembrane Transport ProteinsmetabolismModels,BiologicalModels,MolecularMolecular Sequence DataMolecular StructureProtein ConformationProteinsSerotoninSerotonin Plasma Membrane Transport ProteinsNot in File101110J.Membr.Biol.2132Department of Pharmacology, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520-8066, USA. gary.rudnick@yale.eduPM:17417703J.Membr.Biol.1(20). The structure of LeuTAa is like a shallow 'shot glass', with the opening facing the extracellular space, the base facing the cytoplasm, and the bottom of the 'glass' located in the membrane bilayer. There are 12 TM domains and the leucine binding site is near the middle of the TM 3 and 8. One of the Na+ binding sites is adjucent to the leucine binding site. Crystal structure shows presence of the extracelluar and cytoplasmic gates that are residues and domains of LeuTAa, which can alternately allow access to the binding sites from either side of the membrane bilayer.
Little information is known about the molecular mechanism and structural dynamics of membrane transporters because a crystal structure is static at particular conformational state of the transporter and therefore cannot provide image of complete transport cycle. Further no crystal structures for members of Solute Carrier Family 13, of which NaDC1 is a member, are yet available. Therefore, structural information about NaDC transporters is still lacking.
TRANSLOCATION MECHANISM BY MEMBRANE TRANSPORTERS
Determination of three dimensional structures to atomic resolution has helped to understand various mechanisms of substrate transport across the membrane by membrane transporters ADDIN REFMGR.CITE Sobczak2005830Structural and mechanistic diversity of secondary transportersJournal830Structural and mechanistic diversity of secondary transportersSobczak,I.Lolkema,J.S.2005/4Amino Acid Transport System X-AGBacterial PhysiologyBacterial ProteinsBiological TransportCarrier ProteinsCation Transport ProteinschemistryEscherichia coliEscherichia coli ProteinsMembrane ProteinsMembrane Transport ProteinsMonosaccharide Transport ProteinsMultidrug Resistance-Associated ProteinsphysiologyProteinsSymportersNot in File161167Curr.Opin.Microbiol.82Department of Microbiology, Groningen Biomolecular and Biotechnology Institute, University of Groningen, Kerklaan 30, 9751 NN Haren, The NetherlandsPM:15802247Curr.Opin.Microbiol.1(21). One mechanism of substrate transport, the alternating access mechanism, has been proposed for major facilitator superfamily member transporters such as LacY and GlpT ADDIN REFMGR.CITE Abramson2003825Structure and mechanism of the lactose permease of Escherichia coliJournal825Structure and mechanism of the lactose permease of Escherichia coliAbramson,J.Smirnova,I.Kasho,V.Verner,G.Kaback,H.R.Iwata,S.2003/8/1Amino Acid SubstitutionBinding SitesBiological TransportCell MembranechemistryCrystallizationCrystallography,X-RayenzymologyEscherichia coliEscherichia coli ProteinsgeneticsHydrogen BondingHydrophobicityIon TransportLactoseMembrane Transport ProteinsmetabolismModels,MolecularMonosaccharide Transport ProteinsMutationProtein ConformationProtein Structure,SecondaryProtein Structure,TertiaryProteinsProtonsSubstrate SpecificitySymportersThiogalactosidestransportNot in File610615Science3015633Department of Biological Sciences, Imperial College London, London SW7 2AZ, UKPM:12893935Science1Lemieux2004682The structural basis of substrate translocation by the Escherichia coli glycerol-3-phosphate transporter: a member of the major facilitator superfamilyJournal682The structural basis of substrate translocation by the Escherichia coli glycerol-3-phosphate transporter: a member of the major facilitator superfamilyLemieux,M.J.Huang,Y.Wang,D.N.2004/8Escherichia coliNot in File405412Curr.Opin.Struct.Biol.144Skirball Institute of Biomolecular Medicine and Department of Cell Biology, New York University School of Medicine, 540 First Avenue, New York, New York 10016, USAPM:15313233Curr.Opin.Struct.Biol.1(12;15). In this mechanism, substrate and ions bind to specific binding sites in the transporter. A conformational change takes place in the transporter, the cotransporter reorients itself so that the substrate and ion binding sites are now accessible from another side of the membrane and release of substrate takes place. The empty transporter returns to its original conformational state for the next round of transport. Therefore, the alternating access model not only allows solutes to be transported from one side of the membrane to the other but also provides a mechanism for a transmembrane concentration difference of one solute to be utilized as a driving force to generate a concentration difference for another solute. The serotonin transporter (SERT) also follows an alternating access model which selectively transports 5-hydroxytryptamine (5-HT) into nerve cells togeth e r w i t h N a + a n d C l " a n d t r a n s p o r t s K + i o n o u t o f t h e c e l l A D D I N R E F M G R . C I T E <