Communication
NMR experiments on the transient interaction of the intrinsically disordered N-terminal peptide of cystathionine-β-synthase with heme

https://doi.org/10.1016/j.jmr.2019.07.048Get rights and content

Highlights

  • Improved mapping of transient heme binding to intrinsically disordered proteins.

  • HCBCACON allows to employ prolines as additional probes in interaction mapping.

  • Residue-specific mapping of heme-protein interactions at physiological conditions.

  • Unraveling the heme interaction of a segment of human cystathionine-β-synthase.

Abstract

The N-terminal segment of human cystathionine-β-synthase (CBS(1–40)) constitutes an intrinsically disordered protein stretch that transiently interacts with heme. We illustrate that the HCBCACON experimental protocol provides an efficient alternative approach for probing transient interactions of intrinsically disordered proteins with heme in situations where the applicability of the conventional [1H, 15N]-HSQC experiment may be limited. This experiment starting with the excitation of protein side chain protons delivers information about the proline residues and thereby makes it possible to use these residues in interaction mapping experiments. Employing this approach in conjunction with site-specific mutation we show that transient heme binding is mediated by the Cys15-Pro16 motif of CBS(1–40).

Introduction

Intrinsically disordered proteins (IDP) and protein regions (IDPR) attached to folded protein domains have been found to be implicated in critical biological functions and in neurodegenerative and other diseases [1], [2], [3], [4], [5]. In contrast to covalent binding, as in many metalloproteins, IDPs have been shown to be involved in functionally important transient interactions. In many cases, heme-binding (HBM) or heme-regulatory motifs (HRM) with special amino acid combinations such as CxxHx18H, CxxHx16H and cysteine-proline (CP) in small sequence stretches are responsible for the heme association with moderate binding constants [6], [7], [8], [9], [10]. Coupled with mutagenesis, the heme-protein interaction studies are often carried out via techniques such as resonance Raman, surface plasmon resonance, NMR, circular dichroism (CD) and UV–Vis spectroscopy [11], [12], [13]. Such studies demonstrated the possibility for the IDPR to undergo conformational changes upon heme binding, e.g. due to hexa-coordination of the iron moiety in the iron-porphyrin complex with cysteine and histidine residues located at different positions in the protein sequence, leading to a reduction in the flexibility of the polypeptide chain [8], [9], [10]. A variety of techniques like chemical shift perturbation, relaxation dispersion spectroscopy and paramagnetic relaxation enhancement have been demonstrated for studies of protein-ligand interactions [14], [15], [16], [17], [18] and [1H, 15N]-HSQC experiments are often employed to map the interaction interface. However, in experimental situations leading to fast amide proton exchange with water molecules, e.g. at physiological temperature and pH, its applicability may be limited [19]. Thus, the required protocol strongly depends on the given experimental situation and the use of 13C direct detection experiments [20], [21], [22], [23] has been shown to offer an efficient alternative approach for structural investigations of uniformly enriched proteins and their complexes.

Many IDPs are involved in regulatory interactions with heme, with Cys-Pro (CP) motifs often playing an important role [8], [9], [10]. The proline residue in the CP motif assists the coordination of cysteine thiolate to the Fe(III) heme complex [12], [24]. We are currently investigating the N-terminal peptide stretch (1–40) of human cystathionine-β-synthase (CBS, 551 a.a.; UniProtKB P35520) in the context of heme-IDP interaction [25], [26], [27]. In the enzyme CBS heme is bound as a cofactor via an axial coordination to cysteine-52 and histidine-65. However, NMR and UV/Vis studies [28] have shown that the disordered N-terminal region of CBS also contributes heme-binding capacities via a second binding site, the CP-based motif around cysteine-15 possibly extending to histidine-22. Functional assays revealed that this second heme-binding site increases the efficacy of the enzyme by ∼30%.

Taking into consideration these factors, we aimed at a further robust strategy for probing the transient interaction of the N-terminal peptide stretch of human cystathionine-β-synthase (CBS(1–40)) with heme.

Section snippets

Materials and methods

CBS(1–40) (0.6 mM) was expressed via a fusion protein approach with the streptococcal protein GB1. The sample preparation followed protocols described earlier [29]. Heme was dissolved 1 mM in 20 mM NaOH and incubated for 30 min. NMR measurements on CBS(1–40) were performed in sodium phosphate buffer pH 6.9 and carried out on a Bruker 600 MHz narrow-bore Avance III NMR spectrometer equipped with pulse field gradient accessories, pulse shaping units and triple resonance cryo-probe with the sample

Results

Initial heme-binding studies were carried out via [1H, 15N]-HSQC experiments. To improve the solubility of the target protein, make use of easy purification protocols, and minimize proteolytic degradations, GB1 fusion peptide samples of the N-terminal (1–40) of CBS (wild type and mutants) were employed in these investigations, and it is seen that the N-terminal peptide upon heme binding undergoes transient formation of a hexa-coordinated complex that is sparsely populated and in exchange with

Discussion and conclusions

The objective of this study was to evaluate the heme-binding of the non-crystallizable N-terminal peptide stretch (1–40) of human cystathionine-β-synthase, an intrinsically disordered protein region. As the conventional approach via [1H, 15N]-HSQC interaction mapping experiments was hampered by fast HN exchange, the HCBCACON protocol was adapted as an alternative strategy. This experiment has the combined advantages of being HN exchange-independent and of adding proline correlations as

Compliance with ethical standards

Funding: This work was supported by the Deutsche Forschungsgemeinschaft (DFG) [grant number: FOR 1738 (OH 86/3-2)].

Declaration of Competing Interest

The authors declared that there is no conflict of interest.

Acknowledgments

We thank S. Häfner (CS Protein production, FLI) for technical support. Financial support by the Deutsche Forschungsgemeinschaft (DFG) within FOR 1738 (to D.I. and O.O.) is gratefully acknowledged. The FLI is a member of the Leibniz Association (WGL) and is financially supported by the Federal Government of Germany and the State of Thuringia.

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