Structures of the Escherichia coli transcription activator and regulator of diauxie, XylR: an AraC DNA-binding family member with a LacI/GalR ligand-binding domain

Abstract

Escherichia coli can rapidly switch to the metabolism of l-arabinose and d-xylose in the absence of its preferred carbon source, glucose, in a process called carbon catabolite repression. Transcription of the genes required for l-arabinose and d-xylose consumption is regulated by the sugar-responsive transcription factors, AraC and XylR. E. coli represents a promising candidate for biofuel production through the metabolism of hemicellulose, which is composed of d-xylose and l-arabinose. Understanding the l-arabinose/d-xylose regulatory network is key for such biocatalyst development. Unlike AraC, which is a well-studied protein, little is known about XylR. To gain insight into XylR function, we performed biochemical and structural studies. XylR contains a C-terminal AraC-like domain. However, its N-terminal d-xylose-binding domain contains a periplasmic-binding protein (PBP) fold with structural homology to LacI/GalR transcription regulators. Like LacI/GalR proteins, the XylR PBP domain mediates dimerization. However, unlike LacI/GalR proteins, which dimerize in a parallel, side-to-side manner, XylR PBP dimers are antiparallel. Strikingly, d-xylose binding to this domain results in a helix to strand transition at the dimer interface that reorients both DNA-binding domains, allowing them to bind and loop distant operator sites. Thus, the combined data reveal the ligand-induced activation mechanism of a new family of DNA-binding proteins.

Figure 1.

Structure of E. coli XylR defines a new DNA-binding family. (A) Overall structure of a XylR subunit. α-helices and β-strands are coloured red and yellow, respectively, and labelled, and loops are coloured green. The bound d-xylose is shown as cpk, with carbons and oxygens coloured cyan and red, respectively. The DNA-binding domain and PBP subdomains 1 and 2 are labelled. (B) Topology diagram of the XylR subunit with α-helices and β-strands coloured as in Figure 1A. The residues contained within each secondary structural element are also indicated. The asterisk indicates the region, which encompasses residues 221–229, which is a helix in the apo form and a strand in the d-xylose bound form (the latter of which is shown here). (C) Two views of the XylR dimer (rotated by 90°). One subunit is coloured as in Figure 1A and other is coloured dark blue. (D) Electrostatic surface representation of the XylR dimer (shown in the same orientations as Figure 1C). Electropositive and electronegative regions are coloured blue and red, respectively. This Figure, Figures 2A–B, 3A–B and 4F were made with PyMOL (26).

Figure 2.

d-xylose binding by XylR. (A) Close up of the d-xylose–XylR interaction. XylR residues that make key interactions with d-xylose are shown as sticks and labelled. (B) Comparison of XylR–d-xylose complex with a model of a XylR–l-arabinose complex. As indicated by the transparent surface representations, l-arabinose binding in this mode would result in significant clash with Trp135. (C) ITC studies on d-xylose (left) and l-arabinose (right) binding to XylR. The binding isotherm of XylR for d-xylose resulted in a K_d of 3.3 ± 0.5 uM with a stoichiometry of 1 XylR: 1 d-xylose. By contrast, l-arabinose showed no binding by XylR.

Figure 3.

d-xylose binding triggers helix to strand transition. (A) Superimposition of the apo (green) and d-xylose bound (blue) XylR structure showing a close up of the region undergoing a helix to coil transition. The overlay indicates that d-xylose triggers this response by forcing Asp219 and the accompanying N-terminal region of the helix to move, which requires the helix to unfold. (B) d-xylose binding leads to a helix to strand transition. For clarity the strand is shown as a thin ribbon in this Figure. (C) Overall result of the helix to strand transition upon d-xylose binding is a reorientation of the DNA-binding domains. d-xylose is shown as cpk, the d-xylose binding domain as transparent surfaces and the DNA-binding domains as ribbons.

Figure 4.

XylR DNA binding and activation by d-xylose. (A) Top shows the organization of the two operons regulated by XylR. Below, sequences of the xyl promoters (IA and IF), which are transcribed in opposite directions. The arrows represent the 5′ to 3′ directions of the sequence motifs. (B) The binding affinity of XylR for the IA promoter. The resulting isotherm revealed a binding affinity of ∼33 nM, in the presence of d-xylose. In the absence of d-xylose, no significant binding is observed. (C) The XylR-IF promoter binding isotherm reveals an affinity of ∼25 nM in the presence of d-xylose, whereas in the absence of d-Xylose no binding is observed (monomer concentration). (D) Stoichiometric FP experiment carried out in the same manner as that shown in (C) with the exception that the IF DNA concentration was increased to 1 µM. This concentration is 40-fold higher than the K_d, thus ensuring stoichiometric binding. The transition from high- to low-affinity binding resulted in an inflection point of ∼1 uM XylR in the presence of 1 uM IF DNA. This indicated a binding stoichiometry of one XylR dimer to two DNA duplexes. (E) Model of XylR-operator DNA based on the stoichiometry study in (D) showing two duplexes binding a dimer.

Figure 5.

The binding mode of XylR to the xyl promoter region observed by atomic force microscopy. (A) Schematic of the xyl promoter region, which contain two XylR binding sites (IA and IF). The arrow indicates the 5′ to 3′ direction of the XylR-binding site. (B) A cartoon representation of the two observed modes of XylR-DNA binding. The AFM data show that a XylR dimer binds first to one DNA site and, subsequently, the second site on the same DNA strand, looping the DNA. (C) AFM images of a XylR dimer bound to a single promoter site of the xyl promoter region. (D) A XylR dimer binding to both promoter sites creating a DNA loop. However, the intervening DNA between operator sites was too short to readily visualize via AFM using this DNA site. See Figure 5G. (E) Unnatural DNA substrate used to visualize DNA loop more clearly. (F) AFM images of a XylR dimer bound to a single promoter site of the longer DNA substrate shown in E. (G) AFM images of a XylR dimer binding to the two promoter sites, leading to clear DNA looping.