Protein Complexes Form a Basis for Complex Hybrid Incompatibility

Abstract

Proteins are the workhorses of the cell and execute many of their functions by interacting with other proteins forming protein complexes. Multi-protein complexes are an admixture of subunits, change their interaction partners, and modulate their functions and cellular physiology in response to environmental changes. When two species mate, the hybrid offspring are usually inviable or sterile because of large-scale differences in the genetic makeup between the two parents causing incompatible genetic interactions. Such reciprocal-sign epistasis between inter-specific alleles is not limited to incompatible interactions between just one gene pair; and, usually involves multiple genes. Many of these multi-locus incompatibilities show visible defects, only in the presence of all the interactions, making it hard to characterize. Understanding the dynamics of protein-protein interactions (PPIs) leading to multi-protein complexes is better suited to characterize multi-locus incompatibilities, compared to studying them with traditional approaches of genetics and molecular biology. The advances in omics technologies, which includes genomics, transcriptomics, and proteomics can help achieve this end. This is especially relevant when studying non-model organisms. Here, we discuss the recent progress in the understanding of hybrid genetic incompatibility; omics technologies, and how together they have helped in characterizing protein complexes and in turn multi-locus incompatibilities. We also review advances in bioinformatic techniques suitable for this purpose and propose directions for leveraging the knowledge gained from model-organisms to identify genetic incompatibilities in non-model organisms.

Keywords: bioinformatics; evolution; hybrid incompatibility; proteins; proteomics; speciation.

Figure 1

Protein complex micro-environments provide a molecular basis for multi-locus incompatibilities. Among biomolecular physical interactions within cells that give rise to hybrid incompatibilities, protein-protein interactions (PPIs) are a rich target for experimental investigation. (A) An example of a mutation on a monomeric protein verses a similar mutation at the interface of a PPI. A monomeric protein (magenta) undergoes a mutation at a surface residue from a positively charged amino acid residue to a negatively charged one which displays no phenotype and is fully functional. By contrast, in a protein-protein complex (green-blue) a mutation in one of the subunits (green) at a surface amino acid residue within the interaction domain from a positively charged residue to a negatively charged one could cause a loss of function, which may display a phenotype subject to selective pressure. (B) The mutation in the monomeric protein may be lost over time due to genetic drift. By contrast, among the subunits of the non-functional protein-protein complex, under severe selective pressure, a compensatory mutation may occur in the binding partner protein (blue) to allow for the formation of a function complex where both mutations rapidly fix within the population. However, note that the charge residues on the surface binding sites have been reversed between the subunits. (C) An example of a mild mutation within a protein-protein interaction site. An interaction between subunits in a multi-protein complex are stabilized by non-covalent bonds, where in this example three hydrogen bonds (red) create the binding energy to stabilize the complex. The fictional mutation illustrated here leads to the loss of one hydrogen bond in the protein-protein interaction domain of the complex. Within the cell, a mild mutation may lead to a small decrease in the average stability of the protein complex, illustrated in this diagram as a failure of only a few protein complexes to maintain their structure, yielding a very mild phenotype. Under selective pressure over time, compensatory mutations can occur in the binding partner to suppress the mild defect and fix within the population (bottom), illustrated here as a Van der Waals potential (black). (D) Co-evolved binding partner alleles can be uncovered as incipient mild hybrid incompatibility, which may or may not give rise to a detectable phenotype in hybrids. Even if there is no detectible phenotype, at the quantitative level among the population of individual proteins and protein complexes within the hybrid cells, this incipient incompatibility can lead to failures in the proper assembly of the protein complex and/or lead to a decrease in the stability of the protein complex (bottom). (E) After multiple rounds of mutations that display a mild phenotype, followed by compensatory suppressor mutations in the binding partner, multiple changes in the micro-environment in the protein-protein interaction domains can accumulate and fix in the population. (F) The fixation of these mutations in the protein-protein interaction domains can be revealed later as a source of hybrid incompatibility.

Figure 2

Co-evolving protein-protein interactions depend on the phenotypic traits and stressful environmental and cellular conditions can reveal hybrid incompatibility phenotypes. Illustrated examples of protein micro-environment in a protein-protein complex structural and functional integrity. (A) Large multi-subunit protein complexes are assembled in a step-by-step manner, where hybrid incompatibility may lead to the loss of the entire functional complex, especially if one of the evolved binding partners is necessary for an early assembly step. In this illustration protein 1 and protein 2 must assemble first in order to promote a stable interaction with proteins 3 and 4. If mutation-suppression occurs in divergent organisms, such as the reverse of positively (red) and negatively (green) charged amino acid residues as illustrated here, then a severe disruption in the protein complex assembly can occur (bottom). (B) Under congenial environments, when cells are challenged by mild forms of intrinsic protein-protein incompatibilities, the cellular homeostasis machinery, including chaperones and regulated proteolysis, protects the cells and promotes the formation and selection of functional protein complexes. (C) Even under weak stressful environments (red circle with lightning), mild protein-protein incompatibilities can accentuate to higher levels of unfolded and mis-assembled proteins, leading many cellular complexes to fail and overwhelm the protein homeostasis machinery (gray) causing a collapse in cellular protein homeostasis and proteotoxic stress.

Figure 3

Combining column chromatography with mass spectrometry (MS) can successfully identify protein complexes. The integrity of protein-protein interactions in hybrids can be measured directly using classic protein column chromatography combined with ultra-sensitive MS techniques. (A) Under defined environmental conditions the spectrum of protein complexes in a cell can be characterized by employing a combination of cell extraction, size-exclusion chromatography (gel filtration), and MS analyses on the resultant fractions. Size-exclusion chromatography separates proteins and intact multi-subunit protein complexes based on their size (molecular weight; mw) and shape, where large-sized proteins or complexes elute from the column in early fractions (left), and smaller proteins elute in later fractions (right). Due to the sensitivity of mass spectrometry, even very small perturbations in the physiological states of protein complexes can be detected, even when there is no observable phenotype associated with the protein complex function in the organism (middle). Or, observable mutant phenotypes based on protein-protein interactions that fail can be detected as large bio-signatures of unassembled protein complex subunits (bottom). In this example, only one protein-protein complex is illustrated. In a biological cellular extract sample, there will be 1,000 s of overlapping protein complexes of various sizes and shapes in each fraction, all of which can be detected in unison via mass spectrometry. (B) In hybrids, size exclusion chromatography-mass spectrometry analyses can reveal evidence for weak incipient hybrid incompatibilities that do not display a severe phenotype (bottom). (C) Identifying and mapping the location of protein-protein interactions domains on the surfaces of proteins may identify residues that can contribute to hybrid incompatibilities. Crosslinking reagents chemically react with physically close and exposed amino acid residues on the surfaces of protein binding pairs in a complex (red). These covalent crosslinks are maintained during proteolytic digestion of the proteins with the enzyme trypsin in preparation for mass spectrometry. The cross-linked peptide-peptide fragments will be detected as a single molecule during mass spectrometry (bottom). Analysis of the collection of cross-linked fragments in a sample can be employed to create a physical map of a likely protein-protein interaction domain, a region that will have the potential to contribute to hybrid incompatibility within the potential protein-protein interaction sites in a protein complex.

Figure 4

The importance of advances of bioinformatics tools and algorithms in the field of proteomics and in predicting protein complexes. Non-model organisms can be explored in silico after the completion of genome sequencing and/or transcriptomic analyses with the application of bioinformatics. (A) Databases from model organisms can be employed to make predictions about potential protein complexes in non-model organisms. Based on predicted ORFs from genomic sequencing and/or transcriptomics, orthologs proteins can be identified in non-model organisms and “assembled” in silico to predict the potential existence of a protein complex in the unstudied system. (B) Amino acid residues that have been established or predicted to contribute to hybrid incompatibility in the model organism (represented as red and yellow) are then employed as a target for analyses to investigate the potential for hybrid incompatibility within two related non-model organisms with the potential to make hybrids.