The crystal structure of a liganded trehalose/maltose-binding protein from the hyperthermophilic archaeon Thermococcus litoralis at 1.85 Å

Abstract

We report the crystallization and structure determination at 1.85 A Ê of the extracellular, membrane-anchored trehalose/maltose-binding protein (TMBP) in complex with its substrate trehalose. TMBP is the substrate recognition site of the high-af®nity trehalose/maltose ABC transporter of the hyperthermophilic Archaeon Thermococcus litoralis. In vivo, this protein is anchored to the membrane, presumably via an N-terminal cysteine lipid modi®cation. The crystallized protein was N-terminally truncated, resulting in a soluble protein exhibiting the same binding characteristics as the wild-type protein. The protein shows the characteristic features of a transport-related, substrate-binding protein and is structurally related to the maltose-binding protein (MBP) of Escherichia coli. It consists of two similar lobes, each formed by a parallel b-sheet¯anked by a-helices on both sides. Both are connected by a hinge region consisting of two antiparallel b-strands and an a-helix. As in MBP, the substrate is bound in the cleft between the lobes by hydrogen bonds and hydrophobic interactions. However, compared to maltose binding in MBP, direct hydrogen bonding between the substrate and the protein prevails while apolar contacts are reduced. To elucidate factors contributing to thermostability, we compared TMBP with its mesophilic counterpart MBP and found differences known from similar investigations. Speci®cally, we ®nd helices that are longer than their structurally equivalent counterparts, and fewer internal cavities.

Structural and Thermodynamic Analyses of Solute-binding Protein from Bifidobacterium longum Specific for Core 1 Disaccharide and Lacto-N-biose I

“…trehalose/maltose-binding protein and cyclo/maltodextrin-binding proteins) from other sources have also been determined (33,(45)(46)(47) (49), and liganded open (50) states have been reported. In the case of GL-BP, we could not obtain crystals without ligands even after extensive crystallization screening.…”

Insights into ABC Transport in Archaea

“…In early studies the trehalose-maltose-binding protein (TMBP) was identified, and transport was shown to occur with a very high affinity of 20 nM at 80 • C (Xavier et al, 1996). Later TMBP and MalK, the ATPase domain of this transporter, were crystallized (Diederichs et al, 2000;Diez et al, 2001;see below). Finally, the entire transporter was heterologously expressed in E. coli and purified .…”

Sugar transport in (hyper)thermophilic archaea

“…In the open state, the substrate can interact with the binding site whereupon the two domains come together forming a closed state in which the substrate is occluded. The three-dimensional structure of the catalytic site of both Tl-TMBP (PDB-entry: 1EU8) and Pf-MDBP (PDBentry: 1ELJ) has been solved with bound substrate [7,10]. Although both proteins are structurally similar to the E. coli MalE, some differences can be observed.…”

“…The dissociation constants vary between 40 and 500 nM [1,16] (Table 3). With Tl-TMBP, substrate binding appears to be fast, whereas substrate dissociation is slow and temperature dependent [7,14]. These latter features might be due to the more excessive interactions of the substrate with the binding protein.…”