Paired-Cysteine Scanning Reveals Conformationally Sensitive

Feb 25, 2019 - In contrast, a recent study reports high Hill coeffs. for the activation of .... Canul-Tec, Juan C.; Assal, Reda; Cirri, Erica; Legrand...
0 downloads 0 Views 976KB Size
Download PDF
Subscriber access provided by Washington University | Libraries

Article

Paired-cysteine scanning reveals conformationally sensitive proximity between the TM4b-4c loop and TM8 of the glutamate transporter EAAT1 Wenlong Zhang, Suifen He, Xiuping Zhang, and Shaogang Qu ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.9b00048 • Publication Date (Web): 25 Feb 2019 Downloaded from http://pubs.acs.org on March 2, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 16 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Chemical Neuroscience Conformationally sensitive between TM4 and TM8 of EAAT1

1 2 3

Paired-cysteine scanning reveals conformationally sensitive proximity between the TM4b-4c loop and TM8 of the glutamate transporter EAAT1

4

Wenlong Zhang#1,2 Suifen He#1,2 Xiuping Zhang3 Shaogang Qu*1,2

5 6 7 8 9 10 11 12 13 14 15 16

1Central

Laboratory and Department of Neurology, Shunde Hospital, Southern Medical University (The First

People’s Hospital of Shunde Foshan), Foshan, Guangdong 528300, China. 2Key

Laboratory of Mental Health of the Ministry of Education, Southern Medical University, Guangzhou,

Guangdong 510515, China. 3Teaching

Center of Experimental Medicine, School of Basic Medical Sciences, Southern Medical University,

Guangzhou, Guangdong 510515, China. #Wenlong

Zhang and Suifen He contributed equally to this work.

Running Title: Conformationally sensitive between TM4 and TM8 of EAAT1 *To

whom correspondence should be addressed: Shaogang Qu, Central Laboratory and Department of Neurology,

Shunde Hospital, Southern Medical University (The First People’s Hospital of Shunde Foshan), Foshan, Guangdong 528300, China. E-mail: [email protected]. Voice: +86 757 22819316. Fax: +86 757 22223899

17 18 19 20 21 22 23 24

Author Contributions: Shaogang Qu: Research design; Wenlong Zhang, Suifen He, Xiuping Zhang: Conducted Experiments; Wenlong Zhang, Suifen He: Statistical analyses and manuscript draft; Wenlong Zhang, Suifen He, Xiuping Zhang, Shaogang Qu: Critical revision of the manuscript; all authors: Finalization and approval of the content of the manuscript.

25 26 27 28 29 30 31 32 33 34 35 36 37 38 1

ACS Paragon Plus Environment

ACS Chemical Neuroscience 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 16

Conformationally sensitive between TM4 and TM8 of EAAT1

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

ABSTRACT Excitatory amino acid transporters (EAATs) take up the neurotransmitter glutamate from the synaptic cleft and maintain glutamate concentrations below neurotoxic levels. Recently, the crystal structures of thermostable EAAT1 variants have been reported; however, little is understood regarding the functional mechanism of the transmembrane domain (TM) 4b-4c loop, which contains more than 50 amino acids in mammalian EAATs that are absent in prokaryotic homologs. To explore the spatial position and function of TM4 during the transport cycle, we introduced pairwise cysteine substitutions between the TM4b-4c loop and TM8 in a cysteine-less version of EAAT1, CLEAAT1. We observed pronounced inhibition of transport by Cu (II)(1,10-phenanthroline)3 (CuPh) for doubly substituted V238C/I469C and A243C/I469C variants, but not for corresponding singly substituted CL-EAAT1 or for more than 20 other double-cysteine variants. Dithiothreitol treatment partially restored the uptake activity of the CuPh-treated V238C/I469C and A243C/I469C doubly substituted variants, confirming that the effects of CuPh on these variants were due to the formation of intramolecular disulfide bonds. Glutamate, KCl, and D, L-threo-βbenzyloxy-aspartate weakened CuPh inhibition of the V238C/I469C variant, but only KCl weakened CuPh inhibition of the V243C/I469C variant, suggesting that the TM4b-4c loop and TM8 are separated from each other in the inward-facing conformations of EAAT1. Our results suggest that the TM4b-4c loop and TM8 are positioned in close proximity during the transport cycle and are less closely spaced in the inward-facing conformation. Keywords: TM8, glutamate transporter, TM 4b-4c loop, cross-linking, EAAT1, cysteine scanning

19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43

INTRODUCTION Glutamate is the primary excitatory neurotransmitter in the central nervous system and is recycled from the synaptic cleft by glutamate excitatory amino acid transporters (EAATs), which maintain synaptic communication and prevent neurotoxicity between neurons1, 2. Three sodium ions and one proton along with a substrate are cotransported into the cytoplasm while one potassium ion is counter-transported into the extracellular space3-5. Substrate transport is an electrogenic process generated by Na+-K+-ATPase6-8. Five subtypes of glutamate transporters (EAAT1~EAAT5) share approximately 50% amino acid sequence identity9-14. In particular, EAAT1 and EAAT2 are responsible for recycling glutamate in the rodent brain15. The crystal structure of the glutamate transporter homologue GltPh from Pyrococcus horikoshii is available16. GltPh has been shown to have a trimer structure, with each monomer acting as an independent transporter unit17-20. The monomer consists of eight transmembrane segments (TM1~8) and two reverse hairpin loops (HP1~HP2)17-20. HP1 has been demonstrated to form the intracellular gate, while HP2 participates in the extracellular gating of the substrate16, 21-26. There are two domains on each monomer: the scaffold domain (ScaD), consisting of transmembrane segments TM1~TM2 and TM4~TM5; and the transport domain (TranD), consisting of TM3, TM6~TM8 and reentrant helical loops 1~222, 27, 28. The structure of eukaryotic glutamate transporters is similar to that of prokaryotic ones17-20, although there is only about 35% sequence homology between bacterial and mammalian transporters16,

29.

In particular, a region of

difference is found for TM4, which is composed of TM4a, TM4b and TM4c: there are about 50 amino acids extra on the TM4b-4c loop of eukaryotic EAATs compared to prokaryotic EAATs (Fig. 1A)16. Although crystallization of thermostable EAAT1 has been achieved with a substrate or the allosteric inhibitor UCPH101, most residues in the TM4b–c loop could not be modeled23. Therefore, the molecular mechanism and spatial location of the TM4b-4c loop are unclear. Our previous study identified a complex spatial relationship between transmembrane segment TM2 and TM4 during the transporter cycle by introducing pairwise cysteine substitutions in EAAT230.TM2 is an integral part of 2

ACS Paragon Plus Environment

Page 3 of 16 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Chemical Neuroscience Conformationally sensitive between TM4 and TM8 of EAAT1

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

the scaffold domain and is presumed to maintain the balance of the transporter during glutamate transport16, 23. Because TM2 is relatively static during the transport process, we speculated that TM4 may participate in the translocation of substrate. However, the molecular mechanisms and functions of the TM4b-4c loop during glutamate transport cycle are as of yet unknown. The recent crystal structure of thermostable EAAT1 (EAATcryst) shows that TM8 can be divided into three parts: extracellular (TM8a), transmembrane (TM8b) and cytoplasmic (TM8c) helices respectively (Fig. 1B)23. TM8a plays an important role in the transporter, as evidenced by the pronounced decrease in the transport activity caused by cysteine mutations in TM8a of EAAT131. TM8b is important to the correct folding and function of the transporters23, and on the cytoplasmic side, TM8c interacts with TM3 and TM7a and deletion mutants of TM8c have deleterious effects on transport function and membrane trafficking23, 32. Despite the wealth of information available on TM8 structure and function, how the TM4b-4c loop is positioned relative to TM8 is unknown. In order to clarify the relative spatial proximity and function of TM4 and TM8 in the transport process, we designed 26 mutated transporters with cysteine pair residues and introduced them into a cysteine-less version of EAAT1 (CL-EAAT1)33. Our results demonstrate that the transport activity of V238C/I469C and A243C/I469C mutants was severely decreased after application of Cu (II)(1,10-phenanthroline)3 (CuPh), a catalyst of disulfide bond formation (Fig. 1C), which suggests that the respective amino acid domains are in close proximity. We also determined the effects on crosslinking when external media were applied to induce outward or inward-facing conformations. Finally, the aqueous accessibility of I469C was explored by applying sulfhydryl reagent. Our results suggest that the spatial relationship between Val-238/Ala-243 in the TM4b-4c loop and Ile-469 in TM8 is altered during the transport cycle.

21 22 23 24 25 26 27 28 29

Fig. 1. Sequence alignment and EAAT transmembrane topology (A), (B) Sequence alignment of the TM4b-4c loop (panel A) and TM8 (panel B) of GltPh and EAAT116, 23. The insertion in eukaryotic transporters between helices 4b and 4c is not included in GltPh and is marked by “----”. The arrows show residue V238 of EAAT1, which is absent in GltPh; and residues A243 and I469 of EAAT1, which align to V151 and T387 of GltPh. (C) Representation of EAAT transmembrane topology16. The red dots show the approximate locations of the following cysteine substitutions: V238, A243, and I469 of EAAT1. RESULTS 3

ACS Paragon Plus Environment

ACS Chemical Neuroscience 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Conformationally sensitive between TM4 and TM8 of EAAT1

1 2 3 4 5 6 7 8 9 10

Cysteine cross-linking at amino acids V238C/I469C and A243C/I469C reduces the transport activity of CLEAAT1 To investigate the function and position of the TM4b-4c loop of EAAT1, we introduced 26 cysteine pairs into CL-EAAT1 on V238C or A243C of TM4 and different amino acids of TM8a or TM8b. The transport activities of mutants V238C/I469C and A243C/I469C were severely reduced when they were exposed to 600 μM CuPh, while the CL-EAAT1 and the other mutants were not inhibited (Fig. 2A). The transport activity decreased as the CuPh concentration increased, and when the concentration was 600 nM, the transport activity was less than 10% (Fig. 2B). The V238C, A243C and I469C single mutants were not affected by CuPh, even when they were co-transfected (Fig. 2C). Since I469 is located in TM8a, we suggest that the TM4b-4c loop is positioned in proximity to TM8a within EAAT1 monomers.

11 12 13 14 15 16 17 18 19 20

Fig. 2. Inhibition of D-[3H]-Asp uptake by CuPh for cysteine mutants V238C/1469C and A243C/1469C. (A) Effect of the disulfide crosslinking agent CuPh on the transport activity of the CL-EAAT1 and 26 different double cysteine mutants was determined by measuring D-[3H]-aspartate uptake; (B) Effect of different concentrations of CuPh on D-[3H]-aspartate uptake of CL-EAAT1, V238C/I469C and A243C/I469C; (C) Effect of 600 nM CuPh on D-[3H]-aspartate uptake in cells expressing CL-EAAT1; single cysteine mutants V238C, A243C and I469C; double cysteine mutants V238C/I469C and A243C/I469C; and cotransfected V238C and I469C or A243C and I469C single mutants. All proteins were expressed in HeLa cells. Cells were treated with CuPh in NaCl solution for 5 min at room temperature, and subsequently D-[3H]-aspartate uptake was assayed. Data represent the percentages of uptake activity after incubation with CuPh relative to the uptake in the absence of CuPh and represent 4

ACS Paragon Plus Environment

Page 4 of 16

Page 5 of 16 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Chemical Neuroscience Conformationally sensitive between TM4 and TM8 of EAAT1

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

as the mean ± S.D. of three different experiments done in triplicate. Values that are significantly different from those of CL-EAAT1 were determined by one-way ANOVA (**P