Dataset Information

Tunnels modulate ligand flux in a heme nitric oxide/oxygen binding (H-NOX) domain.

ABSTRACT: Interior topological features, such as pockets and channels, have evolved in proteins to regulate biological functions by facilitating the diffusion of biomolecules. Decades of research using the globins as model heme proteins have clearly highlighted the importance of gas pockets around the heme in controlling the capture and release of O(2). However, much less is known about how ligand migration contributes to the diverse functions of other heme protein scaffolds. Heme nitric oxide/oxygen binding (H-NOX) domains are a conserved family of gas-sensing heme proteins with a divergent fold that are critical to numerous signaling pathways. Utilizing X-ray crystallography with xenon, a tunnel network has been shown to serve as a molecular pathway for ligand diffusion. Structure-guided mutagenesis results show that the tunnels have unexpected effects on gas-sensing properties in H-NOX domains. The findings provide insights on how the flux of biomolecules through protein scaffolds modulates protein chemistry.

SUBMITTER: Winter MB

PROVIDER: S-EPMC3203760 | biostudies-literature | 2011 Oct

REPOSITORIES: biostudies-literature

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Publications

Tunnels modulate ligand flux in a heme nitric oxide/oxygen binding (H-NOX) domain.

Winter Michael B MB Herzik Mark A MA Kuriyan John J Marletta Michael A MA

Proceedings of the National Academy of Sciences of the United States of America 20111012 43

Interior topological features, such as pockets and channels, have evolved in proteins to regulate biological functions by facilitating the diffusion of biomolecules. Decades of research using the globins as model heme proteins have clearly highlighted the importance of gas pockets around the heme in controlling the capture and release of O(2). However, much less is known about how ligand migration contributes to the diverse functions of other heme protein scaffolds. Heme nitric oxide/oxygen bind ...[more]

PMID: 21997213

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Project description:The gasotransmitter nitric oxide (NO) is a critical endogenous regulator of homeostasis, in major part via the generation of cGMP (cyclic guanosine monophosphate) from GTP (guanosine triphosphate) by NO's main physiological receptor, the soluble guanylate cyclase (sGC). sGC is a heterodimer, composed of an α1 and a β1 subunit, of which the latter contains the heme-nitric oxide/oxygen (H-NOX) domain, responsible for NO recognition, binding and signal initiation. The NO/sGC/cGMP axis is dysfunctional in a variety of diseases, including hypertension and heart failure, especially since oxidative stress results in heme oxidation, sGC unresponsiveness to NO and subsequent degradation. As a central player in this axis, sGC is the focus of intense research efforts aiming to develop therapeutic molecules that enhance its activity. A class of drugs named sGC "activators" aim to replace the oxidized heme of the H-NOX domain, thus stabilizing the enzyme and restoring its activity. Although numerous studies outline the pharmacology and binding behavior of these compounds, the static 3D models available so far do not allow a satisfactory understanding of the structural basis of sGC's activation mechanism by these drugs. Herein, application NMR describes different conformational states during the replacement of the heme by a sGC activators. We show that the two sGC activators (BAY 58-2667 and BAY 60-2770) significantly decrease the conformational plasticity of the recombinant H-NOX protein domain of Nostoc sp. cyanobacterium, rendering it a lot more rigid compared to the heme-occupied H-NOX. NMR methodology also reveals, for the first time, a surprising bi-directional competition between reduced heme and these compounds, pointing to a highly dynamic regulation of the H-NOX domain. This competitive, bi-directional mode of interaction is also confirmed by monitoring cGMP generation in A7r5 vascular smooth muscle cells by these activators. We show that, surprisingly, heme's redox state impacts differently the bioactivity of these two structurally similar compounds. In all, by NMR-based and functional approaches we contribute unique experimental insight into the dynamic interaction of sGC activators with the H-NOX domain and its dependence on the heme redox status, with the ultimate goal to permit a better design of such therapeutically important molecules.

Project description:SignificanceLigand selectivity for dioxygen (O(2)), carbon monoxide (CO), and nitric oxide (NO) is critical for signal transduction and is tailored specifically for each heme-protein sensor. Key NO sensors, such as soluble guanylyl cyclase (sGC), specifically recognized low levels of NO and achieve a total O(2) exclusion. Several mechanisms have been proposed to explain the O(2) insensitivity, including lack of a hydrogen bond donor and negative electrostatic fields to selectively destabilize bound O(2), distal steric hindrance of all bound ligands to the heme iron, and restriction of in-plane movements of the iron atom.Recent advancesCrystallographic structures of the gas sensors, Thermoanaerobacter tengcongensis heme-nitric oxide/oxygen-binding domain (Tt H-NOX(1)) or Nostoc puntiforme (Ns) H-NOX, and measurements of O(2) binding to site-specific mutants of Tt H-NOX and the truncated β subunit of sGC suggest the need for a H-bonding donor to facilitate O(2) binding.Critical issuesHowever, the O(2) insensitivity of full length sGC with a site-specific replacement of isoleucine by a tyrosine on residue 145 and the very slow autooxidation of Ns H-NOX and cytochrome c' suggest that more complex mechanisms have evolved to exclude O(2) but retain high affinity NO binding. A combined graphical analysis of ligand binding data for libraries of heme sensors, globins, and model heme shows that the NO sensors dramatically inhibit the formation of six-coordinated NO, CO, and O(2) complexes by direct distal steric hindrance (cyt c'), proximal constraints of in-plane iron movement (sGC), or combinations of both following a sliding scale rule. High affinity NO binding in H-NOX proteins is achieved by multiple NO binding steps that produce a high affinity five-coordinate NO complex, a mechanism that also prevents NO dioxygenation.Future directionsKnowledge advanced by further extensive test of this "sliding scale rule" hypothesis should be valuable in guiding novel designs for heme based sensors.

Dataset Information

Tunnels modulate ligand flux in a heme nitric oxide/oxygen binding (H-NOX) domain.

Publications

Tunnels modulate ligand flux in a heme nitric oxide/oxygen binding (H-NOX) domain.

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