3.5 │ Structural and kinetic properties of complexes created by histone H1 subtypes and interacting protein
In order to determine the types of complexes formed by histone H1 subtypes with interacting partners, the kinetic and structural parameters of binary interactions were evaluated. They include twelve proteins indicated in the APID database as displaying physical association with histone H1 subtypes, also annotated in the BioGRID and IntAct database (Figure 5). To discriminate a type of formed complexes, an occurrence of amino acid residues in the contact binding sites and its location in the secondary structure motifs were analyzed using BIPSPI and GOR method, respectively. Due to that the contact between proteins is reflected by increased proportion of differently structured amino acids 39, the interface residues were grouped according to the biochemical and structural properties of side chain into hydrophobic (non-polar - Ala, Ile, Leu, Met, Phe, Pro, Trp, Val), polar (uncharged - Asn, Cys, Gly, Gln, Ser, Thr, Tyr) and charged (Asp, Arg, Glu, His, Lys). As it is seen in the Figure 5, in all interfaces of histone H1 subtypes prevailed are amino acid residues with charged (Arg, Glu and Lys) and polar (Asn, Gly, Ser and Thr) side chain. Only one amino acid containing a hydrophobic property is a residue of Ala that occur at a ratio amounting from 0.11 to 0.25 to the other residues. Thus, histone H1 subtypes favor the polar and charged interactions, mostly found in non-obligate 40 and usually transient41 complexes. Because the transient complexes are weak associated and poorly optimized 2, they might have at most a few hydrophobic residues which generally predominated in the obligate interaction. 42 A specificity of H1 interactions is likely determined by large residues of Arg, Glu and Lys that make a side chain to side chain contact, compared to other residues, as Ser and Thr, which favor main chain to side chain contact.43 A complement of amino acid residues in the interfaces of H1-interacting proteins is essentially similar. For between protein-protein contact responsible are charged and polar residues, mainly Arg, Glu, Lys, Ser and Thr, which in a 75% of interactions are the sole residues of the interface. Occasionally, a residue of hydrophobic Leu and Met supplements an interacting surface. Such a transient type of histone H1-partner protein interaction inferred from a character of the interfaces residues was confirmed by determination of their positioning in the secondary structure elements. Whereas the regular secondary structures, i.e. α-helix and β-sheets, are stabilized by hydrogen bonds between backbone atoms in not sequence specific mode, they are less important in formation the transient protein-protein interfaces. 44 Compared to them, the non-regular elements, i.e. coils, loops and turns, are enriched in the transient interactions and specify this type of protein complex.41,42 However, both helical and non-regular secondary structures participating in various proportions in the transient interactions 42 may form the mixed motifs characteristic of heterodimeric interfaces. 45 The contact sites of histone H1 subtypes and their partnering proteins are completely devoid of β-sheets but participation of α-helices is frequently observed. Although some of the binding sites are more abundant in the α-helix fraction, the overall number of non-regular elements is higher. They occur in a 63.9% and 62.26% in the interfaces of histone H1 subtypes and their interacting proteins, respectively (Figure 6). In terms of the binding affinity, transient complexes can display a weak and strong interactions, commonly measured by difference in their equilibrium dissociation constant (Kd).46 To define a strength of histone H1 subtypes-partner protein interactions, the binding affinity of emerging complexes was determined using the ISLAND predictor. The resulted values of Kd for all complexes stay in the nanomolar range, from 10-8 to 10-7 (Table 7). According to the categorization of the binding affinities proposed by Veresov47, the complexes created by histone H1 subtypes should be classified as medium – strong transient, for which Kd takes a value between 10-8 and 10-6. Thus, histone H1 subtypes together with partner proteins fall in the transient interactions by forming a medium unstable complexes. They are capable of temporal association and dissociation in dependence on the effects caused by specific stabilizers and triggers, respectively, that determine a characteristic conformational state.48