Changes in quaternary structure in the signaling mechanisms of PAS domains

Abstract

FixL from Bradyrhizobium japonicum is a PAS sensor protein in which two PAS domains covalently linked to a histidine kinase domain are responsible for regulating nitrogen fixation in an oxygen-dependent manner. The more C-terminal PAS domain, denoted bjFixLH, contains a heme cofactor that binds diatomic molecules such as carbon monoxide and oxygen and regulates the activity of the FixL histidine kinase as part of a two-component signaling system. We present the structures of ferric, deoxy, and carbon monoxide-bound bjFixLH in a new space group ( P1) and at resolutions (1.5-1.8 A) higher than the resolutions of those previously obtained. Interestingly, bjFixLH can form two different dimers (in P1 and R32 crystal forms) in the same crystallization solution, where the monomers in one dimer are rotated approximately 175 degrees relative to the second. This suggests that PAS monomers are plastic and that two quite distinct quaternary structures are closely similar in free energy. We use screw rotation analysis to carry out a quantitative pairwise comparison of PAS quaternary structures, which identifies five different relative orientations adopted by isolated PAS monomers. We conclude that PAS monomer arrangement is context-dependent and could differ depending on whether the PAS domains are isolated or are part of a full-length protein. Structurally homologous residues comprise a conserved dimer interface. Using network analysis, we find that the architecture of the PAS dimer interface is continuous rather than modular; the network of residues comprising the interface is strongly connected. A continuous dimer interface is consistent with the low dimer-monomer dissociation equilibrium constant. Finally, we quantitate quaternary structural changes induced by carbon monoxide binding to a bjFixLH dimer, in which monomers rotate by up to approximately 2 degrees relative to each other. We relate these changes to those in other dimeric PAS domains and discuss the role of quaternary structural changes in the signaling mechanisms of PAS sensor proteins.

Figure 1. Ribbon diagram of the structure of deoxy bjFixLH in the P1 crystal form

P1AB and P1CD dimers are shown in yellow and blue, respectively. Secondary structure elements are labeled on the P1B monomer.

Figure 2. Intra- and inter-unit cell contacts between bjFixLH dimers

The number of contacts made by residues in each monomer of (A) P1AB, (B) P1CD, (C) C2AB, (D) R32, (E) ecDOSH, (F) smFixLH, (G) bsYtvA-LOV, (H) avNifL, and (I) crPhot-LOV1 are colored according to the color scale bar. Conserved dimer interface residues are labeled by residue type and number.

Figure 3. Structure-based sequence alignment of PAS domains

PAS sensor domain sequences were aligned using Dali (59) then manually adjusted to best align secondary structure elements. Residues that have greater than 50% of their surface area buried (as calculated using PISA) at the dimer interface are denoted in red. Residues in italics indicate those for which the structure is unknown. Purple bars and asterisks indicate residues in the conserved dimer interface. Non-crystallographic dimers: bjFixLH (P1AB, P1CD, and C2), ecDOSH, bsYtvA-LOV and avNifL. Crystallographic dimers: bjFixLH (R32), smFixLH, and crPhot-LOV1.

Figure 4. Contact map for the intra-dimer interface of bjFixLH dimers

The Cα-atoms for residues making specific contacts with residues within the same or neighboring monomer were mapped onto their respective ribbon structures and are shown as spheres. Cα-atoms from chain A of P1AB, C2AB, and R32 and from chain C of P1CD are shown as blue spheres. Cα-atoms from chain B of P1AB, C2AB, and R32 and from chain D of P1CD are shown as yellow spheres. Contacts are shown between two side-chain atoms (solid line), between a side-chain and main chain atom (dottedline) and between two residues involving both side chain-side chain and side chain-main chain interactions (thick solid line). Conserved dimer interface residues are underlined.