Transient Homodimer Interactions Studied Using the Electron Self-exchange Reaction

Abstract

Protein-protein interactions are vital to cellular function and recent studies have revealed the complexity of protein interaction networks (1, 2). Considering the crowded environment of the cell, weak (transient) protein interactions are expected to be ubiquitous (3). However, given their low affinity, such complexes tend to be overlooked by high-throughput proteomics.The structural features of protein-protein interfaces that enable transient interactions are not well understood, although poor geometric complementarity seems to be a prerequisite (4 -7). The interfaces of electron transfer (ET) 1 protein complexes, which represent an extreme in terms of low affinity and specificity (5), have the poorest surface complementarity (6).Electron self-exchange (ESE) is a simple reaction exhibited by redox proteins that provides a measure of their ET capabilities. An essential step in ESE is homodimer formation giving rise to transient protein interactions on the submillisecond time scale. To investigate the features of transient proteinprotein interactions involved in ET we have studied the influence of ionic strength on the ESE reactivity of a single family of redox metalloproteins. For this purpose we have chosen the plastocyanins (PCus), which are cupredoxins involved in photosynthetic ET, as their surface features vary considerably depending upon the organism from which the protein is obtained, yet their active site structures are almost identical (8).Plastocyanin is one of the best structurally characterized ET metalloproteins and possesses a ␤-barrel structure with the type 1 copper ion buried ϳ6 Å beneath the protein surface (Fig. 1A) (9 -13). Higher plant PCus have two distinct surface features (9, 11), namely the hydrophobic patch surrounding the exposed His 87 ligand, and the acidic patch that is more distant from the copper site (Fig. 1). In the PCu from the fern Dryopteris crassirhizoma (seedless, vascular plant) the acidic region is relocated and surrounds the hydrophobic patch ( Fig. 1) (13). In green algal PCus the acidic patch is more diffuse (12), whereas in the cyanobacterial PCus it is non-existent ( Fig. 1) (10). The charged and hydrophobic patches of PCu are utilized in the interaction with both physiological ET partners; cytochrome f (cytochrome f of the cytochrome b 6 f complex) and photosystem I (PSI) (5,(15)(16)(17)(18)(19)(20)(21). It has been shown that the structure of the interface between PCu and cytochrome f is organism-dependent (5, 19).Herein we study the influence of ionic strength (NaCl) on the ESE rate constants of spinach (higher plant), D. crassirhizoma, Ulva pertusa (green alga), and Anabaena variabilis (cyanobacterium) PCus. We also investigate the influence of the physiologically relevant Mg 2ϩ ion and temperature on the ESE reactivity of spinach PCu, the most highly charged protein studied. Protein modeling and docking simulations have been used to obtain representative structures of the transient homodimers formed. These models highlight the features of the proteinprotein i...

Metalloproteins Containing Cytochrome, Iron–Sulfur, or Copper Redox Centers

“…Only one redox couple is involved, and there is no driving force for this reaction. Measuring electron self-exchange rate constants by NMR provides a more universal way to measure ET transfer activity as it is carried out in T1 copper centers 1241−1249 (reviewed in ref (100)) as well as in other redox centers. 1250−1252 Electron self-exchange rate constants ( k SES ) of T1 copper proteins range from 10 3 to 10 6 M –1 s –1 at moderate to low ionic strength.…”

The ClusPro web server for protein–protein docking

“…As mentioned, we found over 400 publications that reported models constructed by ClusPro. In many applications the models were validated by experimental techniques, including site-directed mutagenesis with NMR, calorimetry, FRET, or surface plasmon resonance 44,82–101 , cross-linking 102–106 , various spectroscopic methods 107–113 , X-ray scattering 114 , electron self-exchange reaction 115 , radiolytic protein footprinting with mass spectrometry 39,40 , hydrogen/deuterium exchange 116 , or intermolecular Nuclear Overhauser Effect restraints 47 . ClusPro results can also be used to design low resolution and hence cost effective validation experiments.…”

Transient Protein Interactions between Cytochrome f and the Phytocyanin Umecyanin

“…The cupredoxins are a family of intermolecular ET proteins that have evolved features that ensure transient interactions 3. 5–8 The most important of these are a flat hydrophobic patch (the “redox‐active face”) surrounding the exposed His ligand of the type 1 (T1) copper site and a collar or cluster of charged side chains (see Figure 1 B and C) 3–5. 7, 8 The importance of these surface patches is demonstrated by the complex (Figure 2 A) formed by the plant photosynthetic partners plastocyanin (PC) and cytochrome f (cyt f ; Figure 1 A).…”

“…Secondly, UMC is devoid of the characteristic hydrophobic patch and therefore lacks the recognition information that normally guides rearrangement of the encounter complex towards the redox‐active conformation. In fact, one side of the UMC β‐sandwich does have a small hydrophobic surface (Val48, Met66, Val71 and Ile73) that participates in the crystal packing interface (∼500 Å 2 , 60 % apolar atoms, i.e., similar in size and composition to the hydrophobic patch surrounding the His ligand found in most cupredoxins8). 13 Therefore, the short‐range forces that come into play in the encounter complex with cyt f might result in a redox‐inactive interaction between the hydrophobic patch surrounding the haem group of cyt f and this cluster of apolar residues on the side of UMC.…”

“…The rugged and polar nature of the redox‐active face (compare Figure 1 B and C) implies that the interaction of UMC with protein partners is different from that described for other cupredoxins 3. 8 This is an exceptional feature of UMC that contrasts with the highly conserved hydrophobic patch found in almost all other cupredoxins.…”