Research paperUsing a collagen heterotrimer to screen for cation-π interactions to stabilize triple helices
Graphical abstract
Introduction
Collagen is the most abundant protein in higher animal. Collagen is composed of three chains forming triple helix. Sequence of each chain is successive triplets Gly-X-Y, where X is often Pro and Y is often (4R)-hydroxyproline (Hyp or O). Except for Pro and Hyp, there are high percentages of Lys and Arg in natural collagen [1], [2]. Lys could form strong salt bridges with Glu and Asp to stabilize collagen [3], [4]. Although the aromatic residues, such as Phe, Tyr, and Trp, are highly disrupting for triple helices [5] and rarely occur in natural collagen sequences, they play essential biological roles. For example, Phe interacts with integrin α2β1 in the motif GFOGER of [6], [7], and CH-π interactions of Pro and Phe could induce higher-order assembly of collagen fibrils [8].
The quadrupole moment of an aromatic ring of Phe/Tyr/Trp could interact with basic residues such as Lys or Arg, which is called cation-π interactions [9]. The cation-π interactions play an essential role in protein stability [10], [11]. Since basic and aromatic residues coexist in collagen [12], a question has been raised whether cation-π interactions could also stabilize collagen.
Recently, some of interchain cation-π interactions in collagen have been characterized with host-guest study, which has further been utilized to induce the formation of heterotrimers and higher-order assembly of collagen [13], [14], [15], [16].
Side chain interactions in collagen could be location- and residue-dependent. There are two major types of interactions i.e. an axial Y to X′ and lateral Y to X position in two adjacent chains of GXYGX′Y′. The switching of residues between Y and X′/X position had significant effects on the strength of salt bridges [4], [17]. The similar situation could occur for cation-π interactions, so a set of systematic experiments should be made to enumerate all possible pairs and dissect two types of interactions.
Due to the high stability of Pro/Hyp in triple helices, homotrimeric sequences such as (POG)n and (PPG)n had long been used as hosts to contain axial and lateral pairs of amino side chains [3], [13], [18]. Increase of the melting temperatures of YGX motifs could indicate axial interactions, while the XYG motifs could show lateral ones. However, there are two major disadvantages of homotrimeric hosts. First, two axial and one lateral interactions simultaneously exist in the YGX motifs. Secondly, when n and m residues are, respectively, at Y and X/X′ positions in the pairs, n × m peptides should be synthesized for the pairwise interactions. In contrast, a single interaction pair could be pinpoint in a heterotrimeric host, since a mutation does not occur cross three chains. For n × m pairwise interactions, only n + m peptides are synthesized and combined into n × m pairs.
In order to characterize side chain interactions in collagen, a heterotrimeric host should ideally have three features. First, the heterotrimer should be the most stable among all the 27 possible association stoichiometries and chain registries. The (POG)10 homotrimer was more stable than the heterotrimer, (POG)10:(EOG)10:(PRG)10, Hartgerink et al. made a pioneering design of heterotrimer, (POG)10:(EOG)10:(PRG)10, of which the component, (POG)10, could form a more stable homotrimer [19]. Secondly, an obligate 1:1:1, rather than 2:1 or 1:2 stoichiometry is required in order to prevent coexistence of other interchain interactions. For example, a heterotrimer (PKG)10:(DKG)10:(EPG)10 designed by Hartgerink et al. could be used [20]. Thirdly, high-resolution structural information should be known in order to clarify the specific chain registry among six possible ones of 1:1:1 association stoichiometry and precisely pinpoint interchain interactions. A recently resolved crystal structure of collagen heterotrimer, abc, could be an ideal platform to characterize the interchain cation-π pairs (PDB ID: 5YAN) [17], [21].
Based on the abc structure, a library was constructed containing 15 peptides with single mutations at three locations, which were combined into double mutations to measure stabilizing effects (ΔΔTm) of 12 axial and 12 lateral cation-π interactions between Phe/Tyr/Trp and Arg/Lys. Occurring frequencies of the cation-π pairs in natural collagen sequences were calculated and correlated to ΔΔTm, which helps us gain insights into the mechanisms of collagen folding.
Section snippets
Peptide synthesis
The peptides were made with a solid-phase FMOC synthesis method, then went through reverse-phase HPLC to obtain the 95% purity of the products, which were verified with mass spectrometry at GL Biochem (Shanghai) Ltd. (www.glbiochem.com). We confirmed molecular weight and purity of the peptides in our laboratory with HPLC and mass spectrometry (Fig. S1). The peptides were uncapped in both N- and C-termini. The first letter of a mutant peptide represents its original peptide, which is followed by
Constructing a mutation library to screen for side chain interactions
Without disrupting the existing salt bridges of heterotrimer, abc, (PDB ID: 5YAN) [17], Pro10 at Y position in chain b could make a lateral interaction with Hyp9 in X position and axial one to Hyp12 at X′ position in chain c (Fig. 1). Each of these three locations was mutated to three aromatic (Phe/Tyr/Trp) and two basic (Arg/Lys) residues, where 15 mutant peptides were synthesized in total. Three aromatic mutants of chain b were mixed with the original chain a and two basic mutants at the X′
Conclusion
Five axial, i.e. FK, YK, WK, RF, and RW, and one lateral, i.e. RF, cation-π pairs between aromatic and basic side chains in triple helices were screened with a heterotrimeric peptide library containing 15 single mutations and 24 double mutations. Statistical analysis showed that axial RF is used most frequently in natural collagen, while the other pairs, such as FK, YK, WK, were rarely found. Except for the cation-π interactions, Arg plays an important role in the binding of integrin α2β1 [6],
Acknowledgement
This work was supported by 1000 Plan of China (K2069999) and NSFC of Jiangsu Province (BK20151126), NSFC (51603089), National First-class Discipline Program of LITE (LITE2018-03) to FX, NSFC (21603088) and NSFC of Jiangsu Province (BK20161066) HNZ.
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