Polyamines: Naturally occurring small molecule modulators of electrostatic protein–protein interactions

https://doi.org/10.1016/j.jinorgbio.2009.10.007Get rights and content

Abstract

Modulations of protein–protein interactions are a key step in regulating protein function, especially in networks. Modulators of these interactions are supposed to be candidates for the development of novel drugs. Here, we describe the role of the small, polycationic and highly abundant natural polyamines that could efficiently bind to charged spots at protein interfaces as modulators of such protein–protein interactions. Using the mitochondrial cytochrome P45011A1 (CYP11A1) electron transfer system as a model, we have analyzed the capability of putrescine, spermidine, and spermine at physiologically relevant concentrations to affect the protein–protein interactions between adrenodoxin reductase (AdR), adrenodoxin (Adx), and CYP11A1. The actions of polyamines on the individual components, on their association/dissociation, on electron transfer, and on substrate conversion were examined. These studies revealed modulating effects of polyamines on distinct interactions and on the entire system in a complex way. Modulation via changed protein–protein interactions appeared plausible from docking experiments that suggested favourable high-affinity binding sites of polyamines (spermine > spermidine > putrescine) at the AdR–Adx interface. Our findings imply for the first time that small endogenous compounds are capable of interfering with distinct components of transient protein complexes and might control protein functions by modulating electrostatic protein–protein interactions.

Introduction

From metabolic pathways to cellular signaling and intercellular communication, most biological processes depend on the transient formation of specific complexes between diverse proteins, thereby enabling a highly organized, economic transfer of energy, charged particles, and of intermediary metabolites. Structural and mutational analyses imply a central role of electrostatic interactions in protein assembly [1], [2]. As the large protein–protein interfaces often show charged spots, it is widely accepted that the specificity of association is governed by intermolecular salt bridges and H-bonds, whereas hydrophobic forces contribute to the energy balance and this way stabilize the protein complex.

Modulation of protein assembly and thereby protein functions offers new opportunities for therapeutic intervention exploiting protein interfaces as important novel targets for small interfering molecules [3], [4]. However, because interfaces lack defined binding pockets, the search for specific small molecule antagonists/agonists is difficult and still in its infancy [5]. Identification of modulators and knowledge of their way of interference with protein interfaces, on the other hand, will give valuable information for the design of new modulators and potential drugs. Considering the nature of protein–protein interactions, we propose a major modulating role of the small, polycationic and highly abundant natural polyamines that might efficiently bind to negatively charged spots at protein interfaces.

Ubiquitously expressed in cells and also present in mitochondria [6], polyamines are involved in fundamental biological processes, including cell proliferation and differentiation [7], [8], [9]. Polyamine levels are tightly controlled by synthesis and degradation, undergo rapid changes during the cell cycle and reach millimolar concentrations in proliferating cells [7], [10]. Except for the prostate gland and the pancreas where polyamine concentrations are higher, polyamine levels in most tissues fall within the range of 0.1–2 μmol g−1. In human prostate cancer cell lines the spermine and spermidine levels are around 6 mM [11], [12], [13], [14]. Because of their high abundance, their flexibility, and the presence of efficient transport systems, the polycationic polyamines can affect structures and function of negatively charged biopolymers throughout the cell and in the extra-cellular space. A large body of data describes the electrostatic interactions of polyamines with nucleic acids, the resulting stabilization of their structures, and the strong impact on various steps of protein biosynthesis [7], [8]. Electrostatic interactions also govern transport and metabolism of polyamines by specific proteins as well as signaling via receptors and ion channels [15], [16].

In earlier phases of CYP-research, several groups have shown effects of polyamines on functions of CYP’s, however, without examining the mechanistic background [17], [18], [19]. To test whether the major polyamines putrescine, spermidine, and spermine [20] (see Supplementary material Fig. SM1) might affect protein–protein interactions, we have chosen the bovine mitochondrial CYP11A1 electron transfer system as an appropriate model. This three-component system catalyses the key step of steroid hormone biosynthesis (sex hormones, glucocorticoids, mineralocorticoids), the oxidative side chain cleavage of cholesterol to pregnenolone. In this multi-step process, molecular oxygen bound to CYP11A1 is activated by electrons derived from NADPH and sequentially transferred via adrenodoxin reductase (AdR, a NADPH-dependent FAD containing reductase) and adrenodoxin (Adx, an iron-sulfur protein) to the terminal oxidase. Steroid hormone biosynthesis mainly takes place in the adrenals and gonads, where significant changes of the polyamine concentration under different conditions were observed (e.g. an up to 10-fold increase during pregnancy) [21].

The CYP11A1 electron transfer system has been studied in detail by NMR spectroscopy, chemical modification of amino acid residues, site-directed mutagenesis and particularly X-ray crystallography. Three-dimensional structures of bovine Adx, AdR and the cross-linked Adx–AdR complex have been solved [22], [23], [24], [25]. All these studies point to electrostatic interactions as the major forces between the components of the CYP11A1 electron transport chain, showing a crucial role of negatively charged residues on Adx in the recognition of corresponding positively charged ones on AdR and CYP11A1 (for review see [26]). The salt dependence observed for all reduction processes and especially for the CYP11A1 reduction step reflects also the electrostatic nature of complex formation between the corresponding redox partners [27].

The present study examines whether the three major polyamines are capable of modifying the essential interactions between Adx and AdR as well as between Adx and CYP11A1, and, thereby, the enzymatic function at physiologically relevant concentrations. A broad set of techniques was exploited, namely spectrophotometric titration, surface plasmon resonance, stopped-flow methods, and a CYP11A1-dependent cholesterol conversion assay. Moreover, docking experiments (using AutoDock 4 [28], [29]) were performed to identify potential binding sites of polyamines at the surfaces of AdR and Adx. The outcome of these studies indicates for the first time a role of natural polyamines in protein–protein interactions, and specifically as potential modulators of the steroid hormone cascade.

Section snippets

Materials

Cholesterol, putrescine, spermidine, and spermine were purchased from Sigma; glucose-6-phosphate (G6P), G6P dehydrogenase from Roche; HEPES from Roth; Tween20 from Merck; NADPH from GERBU, and cholesterol oxidase from Serva. Fresh solutions of polyamines were prepared for each experiment.

Mutagenesis

The AdxD15K and AdxD15N mutants were prepared in the pKKHC-Adx plasmid using the QuikChange site-directed mutagenesis kit (Stratagene, LaJolla, CA). We determined the correct identity of each mutant by

Results

To examine the effects of polyamines on the CYP11A1 system, we used the three major polyamines putrescine, spermidine and spermine (Fig. SM1) in a broad concentration range (μM–mM) that covered physiological levels also in mitochondria [11]. At concentrations above 1 mM, spermidine and spermine (but not putrescine) substantially increased the pH of the 50 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethane-sulfonic acid) buffer containing 0.05% Tween20 (HEPES–Tween test buffer). Consequently, in all

Discussion

There is clear evidence that the components of cytochrome P450 electron transfer systems interact in a coordinated, highly specific manner and that electrostatic forces between only few amino acids – hot spots – at the large interfaces play a crucial role in these interactions [25], [26], [51]. Targeting these sites by small molecules could modulate the interactions between the proteins and thereby the enzymatic function. We report here for the first time that the small endogenous polyamines

Acknowledgements

The authors thank Dr. B. Schiffler for his initial experiments with polyamines, Dr. A. Zöllner for competent advice during stopped-flow and optical biosensor measurements, and K. Bompais and W. Reinle for their excellent advice and expert technical assistance. This work was supported by a Grant from the Deutsche Forschungsgemeinschaft to R.B.

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