Computational Identification of Functional Centers in Complex Proteins: A Step-by-Step Guide With Examples

Abstract

In proteins, functional centers consist of the key amino acids required to perform molecular functions such as catalysis, ligand-binding, hormone- and gas-sensing. These centers are often embedded within complex multi-domain proteins and can perform important cellular signaling functions that enable fine-tuning of temporal and spatial regulation of signaling molecules and networks. To discover hidden functional centers, we have developed a protocol that consists of the following sequential steps. The first is the assembly of a search motif based on the key amino acids in the functional center followed by querying proteomes of interest with the assembled motif. The second consists of a structural assessment of proteins that harbor the motif. This approach, that relies on the application of computational tools for the analysis of data in public repositories and the biological interpretation of the search results, has to-date uncovered several novel functional centers in complex proteins. Here, we use recent examples to describe a step-by-step guide that details the workflow of this approach and supplement with notes, recommendations and cautions to make this protocol robust and widely applicable for the discovery of hidden functional centers.

Brachypodium distachyon ERECTA-like1 protein kinase is a functional guanylyl cyclase

“…A highly conserved and stringent GC search motif for higher plants [KS]X[SCG]X (10)[KR]X(0,3)[DHSE] was constructed, and its continuous refinement in recent years has allowed for the discovery of a number of functional candidate GCs in the plant kingdom [13][14][15][16][17][18][19][20]. Based on bioinformatics prediction of the GCPred tool (http://gcpred.com), the BdERL1 protein was identified as a potential new guanylyl cyclase harboring a conserved GC motif that is localized within the cytosolic C-terminus of the studied protein (BdERL1 GC center 840−853 ) [21].…”

“…1A), where serine at position 1 forms a hydrogen bond with guanine, glycine at position 3 confers substrate specificity for GTP and lysine at position 14 stabilizes the transition state from GTP to cGMP. Additionally, two amino acids away from the C-terminal end of the GC sequence is aspartic acid [D], which is responsible for interacting with Mg 2+ /Mn 2+ ions [7,8,19,20] (Fig. 1B).…”

“…2A). Furthermore, the GC center of BdERL1 contains a characteristic alpha-helix barrel followed by a loop secondary fold that is an innate feature of experimentally validated plant GCs and ACs identified by this motifbased approach [20]. Further probing of the GC center by molecular docking of GTP suggested that GTP can dock at the GC center with a good free energy value (-5.6 kcal/mol) and a favorable binding pose where the negatively charged phosphate end of GTP points toward K853 (position 14 of the motif) while the guanine end is surrounded by negatively charged residues including S840 (position 1 of the motif) (Fig.…”

“…It was previously described in an animal retinal guanylyl cyclase that substitution of the amino acid that confers substrate specificity can convert a GC into an AC [32]. In the case of plant GCs, the amino acid at position 3 of the motif was hypothesized to confer substrate specificity [20,33], and therefore, we mutated this amino acid (G 842 ) in BdERL1 to the negatively charged aspartic acid residue (D) to investigate whether this mutation affects the GC and AC catalytic activities. As previously described, sitedirected mutagenesis by substituting amino acids at the positions responsible for substrate specificity can turn a GC into an AC and vice versa [8,32,34].…”

Functional Crypto-Adenylate Cyclases Operate in Complex Plant Proteins

“…Yet, plant enzymes that generate cAMP and adenylyl cyclases (ACs) were until recently unknown because homologs from bacteria and animal systems appeared to be absent in higher plants. The implementation of an amino acid search motif supported by structural modeling strategy ( Wong et al, 2018 ; Zhou et al, 2021 ) has enabled the discovery of many AC domains. Many of those ACs are somewhat hidden in complex multi-functional proteins ( Al-Younis et al, 2015 ; Chatukuta et al, 2018 ; Bianchet et al, 2019 ; Ruzvidzo et al, 2019 ).…”