Abstract
TrmB is an alpha-glucoside-sensing transcriptional regulator controlling two operons encoding maltose/trehalose and maltodextrin ABC transporters of Pyrococcus furiosus. The crystal structure of an N-terminal truncated derivative of TrmB (amino acids 2-109 deleted; TrmB(delta2-109)) was solved at 1.5 A resolution. This protein has lost its DNA binding domain but has retained its sugar recognition site. The structure represents a novel sugar-binding fold. TrmB(delta2-109) bound maltose, glucose, sucrose, and maltotriose, exhibiting Kd values of 6.8, 25, 34, and 160 microM, respectively. TrmB(delta2-109) behaved as a monomer in dilute buffer solution in contrast to the full-length protein, which is a dimer. Co-crystallization with bound maltose identified a binding site involving seven amino acid residues: Ser229, Asn305, Gly320, Met321, Val324, Ile325, and Glu326. Six of these residues interact with the nonreducing glucosyl residue of maltose. The nonreducing glucosyl residue is shared by all substrates bound to TrmB, suggesting it as a common recognition motif.
Highlights
TrmB is the transcriptional repressor for the gene cluster encoding the trehalose/maltose ABC transporter in hyperthermophilic archaea T. litoralis and P. furiosus [18]
The maltodextrin ABC transporter is distinct in its substrate specificity from the trehalose/maltose ABC transporter, which recognizes maltose and trehalose but not maltodextrins
We found that TrmB controls the mdxE operon of P. furiosus as a transcriptional repressor and showed that repression is abolished by maltotriose and sucrose but not by maltose or trehalose [19]
Summary
Construction of Truncated TrmB Proteins—All truncated TrmB mutants were cloned into pCS19, resulting in C-terminal His tag versions. We transformed the constructed plasmids into Escherichia coli SF120 competent cells and purified the three mutants truncated in the N-terminal DNA binding domain, ⌬2–30, ⌬2–59, and ⌬2–109 TrmB, by Ni2ϩ-NTA2 affinity chromatography. After centrifugation of the precipitated proteins, the supernatant was loaded onto a Ni2ϩ-NTA superflow column from Qiagen equilibrated with buffer I. The supernatant was incubated at 80 °C for 20 min, and after centrifuging for 60 min at 100,000 ϫ g, the supernatant was loaded onto an Ni2ϩ-NTA superflow column from Qiagen equilibrated with running buffer. Crystal Structure Determination and Refinement—The crystallographic phase problem was solved by multiple isomorphous replacement plus anomalous differences using one native data set, one platinum derivative data set, one uranylacetate derivative data set, and one native data set measured at a wavelength of 1.5 Å, allowing the search for the anomalous signal of the sulfur atoms inherent to the protein. Labeled sugar-binding inhibition assays with other sugars (maltose, maltotriose, sucrose, and glucose) were done in the same way with inhibiting sugars present between 25 and 500 M, respectively
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