Structural basis for the hydrolysis of ATP by a nucleotide binding subunit of an amino acid ABC transporter from Thermus thermophilusJournal of Structural Biology


Seenivasan Karthiga Devi, Vishnu Priyanka Reddy Chichili, J. Jeyakanthan, D. Velmurugan, J. Sivaraman
Structural Biology


by he

C enna , Sin

Article history:

Received 2 December 2014

Received in revised form 20 April 2015 (Higgins, 2001). ATP binding and/or hydrolysis of the NBDs is known to be required for the dynamic nature of these ABC transporters (Davidson et al., 2008). o acid ABC involved diates in the amino acid ABC transporter of the gram-negative aerobic eubacterium Thermus thermophilus from the strain HB8.

TTHA1159 is the NBD that binds to and hydrolyzes ATP for amino acid transport. Along with mutational studies, modeling, and in conjunction with the literature, our data suggest that the conserved Gln86, Asp163 and Glu164 residues of TTHA1159 are involved in ATP hydrolysis. ⇑ Corresponding authors.

E-mail addresses: (D. Velmurugan), dbsjayar@nus. (J. Sivaraman). 1 Equally contributing authors (SKD and VPRC).

Journal of Structural Biology xxx (2015) xxx–xxx

Contents lists available at ScienceDirect

Journal of Struc .especificity, and two nucleotide-binding domains (NBDs), which couple the transport of solutes with the hydrolysis of ATP and nitrogen metabolism (Hosie and Poole, 2001). Here, we report the crystal structures of the ATP-binding subunit (TTHA1159) of2009), with the human genome known to have 49 ABC genes divided into 8 subfamilies (Vasiliou et al., 2009). These proteins possess a common molecular architecture of two transmembrane-spanning domains (TMDs), which determine substrate

Ambudkar, 2001).

Among the ABC transporter family, the amin porters play a crucial role in bacteria and are uptake of amino acids, which are key interme 1047-8477/ 2015 Elsevier Inc. All rights reserved.

Please cite this article in press as: Devi, S.K., et al. Structural basis for the hydrolysis of ATP by a nucleotide binding subunit of an amino acid ABC porter from Thermus thermophilus. J. Struct. Biol. (2015), the carbon1. Introduction

ATP binding cassette (ABC) transporters are involved in the active transport of solutes, such as ions, amino acids, peptides, sugars and proteins, across cellular membranes, and are widely conserved across all three kingdoms of life. ABC transporters comprise one of the four major gene families in humans (Rees et al.,

Depending on the direction of transport of the substrate, ABC transporters act as importers or exporters, with importers generally found in prokaryotes, and exporters present in both prokaryotes and eukaryotes. In humans, the importance of ABC transporters is demonstrated in a number of disorders, such as cystic fibrosis, Stargardt macular dystrophy, Tangier disease, and multidrug resistance in tumor cells (Gottesman andAccepted 20 April 2015

Available online xxxx


Thermus thermophilus


Crystal structure



TransportersATP-binding cassette (ABC) transporters are a major family of small molecule transporter proteins, and their deregulation is associated with several diseases, including cancer. Here, we report the crystal structure of the nucleotide binding domain (NBD) of an amino acid ABC transporter from Thermus thermophilus (TTHA1159) in its apo form and as a complex with ADP along with functional studies. TTHA1159 is a putative arginine ABC transporter. The apo-TTHA1159 was crystallized in dimeric form, a hitherto unreported form of an apo NBD. Structural comparison of the apo and ADP-Mg2+ complexes revealed that

Phe14 of TTHA1159 undergoes a significant conformational change to accommodate ADP, and that the bound ADP interacts with the P-loop (Gly40–Thr45). Modeling of ATP-Mg2+:TTHA1159 complex revealed that Gln86 and Glu164 are involved in water–mediated hydrogen bonding contacts and Asp163 in Mg2+ ion–mediated hydrogen bonding contacts with the c-phosphate of ATP, consistent with the findings of other ABC transporters. Mutational studies confirmed the necessity of each of these residues, and a comparison of the apo/ADP Mg2+:TTHA1159 with its ATP-complex model suggests the likelihood of a key conformational change to the Gln86 side chain for ATP hydrolysis.  2015 Elsevier Inc. All rights reserved.a r t i c l e i n f o a b s t r a c tStructure Report

Structural basis for the hydrolysis of ATP of an amino acid ABC transporter from T

Seenivasan Karthiga Devi a,1, Vishnu Priyanka Reddy

J. Sivaraman b,⇑ aCentre of Advanced Study in Crystallography and Biophysics, University of Madras, Ch bDepartment of Biological Sciences, National University of Singapore, Singapore 117543 cDepartment of Bioinformatics, Alagappa University, Karaikudi 630 004, India journal homepage: wwwa nucleotide binding subunit rmus thermophilus hichili b,1, J. Jeyakanthan c, D. Velmurugan a,⇑, i 600 025, India gapore tural Biology lsevier .com/locate /y jsbitransDetailed Materials and Methods are provided in the the a1-helix and is thus sandwiched between clusters of a-helices on one side and predominantly b-strands along with several loops transporter. A search for structurally similar proteins within the 3.3. Dimeric apo–TTHA1159

The in vivo or reconstituted histidine and maltose uptake systems (HisP and MalK) – two widely characterized ABC transporters – indicate the existence of dimer NBDs; however, there is reported difficulty in obtaining stable dimers of individual ABC NBDs in their purified form, even in the presence of nucleotides (Benabdelhak et al., 2005). In the present case, we have observed monomers in size-exclusion chromatography and dimers in crystallization and Dynamic Light Scattering (DLS) experiments (Fig. S2). In the apo dimer, the buried surface area is 5% (593 Å2/monomer) of the total accessible surface area of each TTHA1159 monomer, and the residues Glu172, Glu176 and Arg240 of both monomers are involved in hydrogen bonding contacts and the salt-bridge between Arg240 and Glu176 increases the stability of the dimer (Fig. S3 and Table S2).

Notably, the crystal structure of the dimeric MalK in open (apo) and closed (nucleotide-bound) forms revealed that the 136-residue regulatory domain (RD) at the C-terminus contributes more to the stability of the dimer than the 235-residue nucleotide-binding domain (NBD) at the N-terminus (Chen et al., 2003). Our crystal structure of dimeric apo-TTHA1159 most resembles the open