We, Constantin and Dominik, the newest members of OPIG (SABS rotation students, as usual) were lucky to have a conference suitable to our research within our rotation period and, granted an allowance from the powers that be, were able to visit this year’s Prague Protein Spring with the topic ‘Proteins at Work’. There, we spent four busy but very inspirational days with about 50 participants in a little palace, the Vila Lanna.

The general topic of this meeting led to a broad variety of talks representing a multitude of fields of protein research: from origins of life, over fuzzy intrinsically disordered proteins and crowded cells to metagenomics and functional sequence alignment annotation.

We picked four thought engaging talks to present at the group meeting on 08/05/2018; here are their summaries:

Protein engineering and in vitro evolution studies for the origins of life

Kosuke Fujishima from Tokyo Institute of Technology presented several examples of the research he conducts in the area of origins of life. Research on the origins of life are generally based around the questions how prebiotic monomers were created, how they condensed into polymers and how functionality emerged within these polymers.

The first example of his research deals with the condensation of prebiotic monomers on the ocean-earth crust-interface. Water cycling between the ocean and the outer layers of the earth’s core provided an environment of high pressure and high temperatures (80 – 200 °C) which is necessary for amino acid polymerisation. The mineral Olivine was found to attract amino acids to its surface and the serpentinisation reaction happening with Olivine might provide the necessary wet/dry cycle. Therefore, the researchers built a reactor aiming to investigate this potential polymerisation mechanism. They found that with providing six prebiotic amino acids, 28 out of 36 possible dipeptides could be found in the reactor. Furthermore, up to 10-mer linear polypeptides could be detected as well, providing evidence for a mechanism of early earth’s generation of polypeptides [unpublished].

The second project showed that both enzymes, CysE/CysK, responsible for the current production of cysteine from serine, could be re-engineered to contain no cysteine in their sequence. Interestingly, cysteine-free CysE showed higher reaction rates than the wild type. Additional reduction to cysteine- and methionine-free enzyme sequences only worked for CysE but not for CysK.[Fujishima et al. (2018)] Still, the experiments indicate that an enzyme world could have existed with a reduced number of amino acids compared to the 20(+) amino acids that we know today.

The third project we wanted to point out used a type of mRNA display that not only links the genotype (mRNA) with its corresponding phenotype (translated protein) but also allows the translated protein to interact with a randomised, non-translated part of the mRNA. This provided a framework for investigating the evolution of ribonucleotide-binding (RNP) proteins. When selecting for ATP-binding, it was observed that protein together with RNA had the best fitness landscape compared to protein selection or RNA selection alone. Further analysis revealed that most binding affinity of the ribonucleotide protein stemmed from its RNA part.[unpublished] These results give rise to the suggestion that RNA and proteins co-evolved, opposing the idea of a pure RNA world.

RNA-protein interactions and the structure of the genetic code

The next speaker added more to the research area of RNA-protein interaction and evolution. Bojan Zagrovic from the University of Vienna presented his research around the finding that pyrimidine (PYR) density of RNA regions is correlated with the corresponding protein region’s affinity to pyrimidine-containing bases (running means of 21 amino acids or 63 bases were used), with the highest correlation between mRNA PYR density and guanine affinity, having an average ‘typical’ Pearson correlation coefficient of 0.80.[Polyansky & Zagrovic (2013)]

This correlation is specific for the current genetic code, shown by random generation of genetic codes which could not reproduce such a correlated behaviour and by looking into three organisms with very different codon usage bias (homo sapiens, E. coli, M. jannaschii). Even though the three averages of codon usage were very different, the highest correlating pairs of mRNA and cognate proteins clustered together, having very similar codon usage. This was also true for the worst correlating pairs.[Hlevnjak & Zagrovic (2015)]

But the big question being: what does this correlation imply functionally?

Annotation analysis revealed that the highest correlating pairs were enriched in nucleotide-binding functions and intrinsically disordered proteins. Without claiming generalisability, Professor Zagrovic pointed out a case study done on RNA polymerase II which has a long disordered C-terminus build up by 26 repeats of a 7 amino acid motif. 248 RNAs were found to interact with RNA polymerase II and in all three reading frames of the interacting RNAs, amino acid codons of the polymerase’s C-terminus were enriched.[unpublished]

This indicates some regulation over gene expression but also several other hypotheses were made: the correlation between the protein regions’ affinity for their cognate mRNA regions might be relevant in virus assembly, since coding RNA and translated proteins have to be in close proximity with each other. The same could be true for some non-membrane-bound compartments, e.g. P-bodies. Or is this correlation characteristic a hint to mRNAs acting as chaperones for their respective proteins? The functional implications of this correlation, while highly speculative, nevertheless suggest exciting research to come in the future.

Fuzziness in protein assemblies

Research from a different, but equally thought provoking field was presented by Mónika Fuxreiter from the University of Debrecen. Her talk on the concept of fuzziness in protein complexes, which she introduced 10 years ago [Tompa & Fuxreiter (2008)], shed light on some more recent developments in the field as well as explaining the underlying concept for those of us (ourselves included) who have not encountered the concept as such before.

Fuzziness in the context of protein complexes describes a phenomenon in which intrinsically disordered proteins, instead of folding upon binding as one would usually observe, can sample several conformational states with different propensities, leading to the sampled states contributing with different strengths to the function of the protein complex and further leading to varying degrees of disorder in the bound state.

This observation has several implications for the understanding of the functionality of disordered proteins, since the relative propensity for different ensemble states in the bound form is thought to be highly susceptible to milieu influences, such as tissue specific splicing and post-translational modifications. Fuzziness (a term that was borrowed from the mathematical theory of fuzzy sets) could thus be a driver of functional adaptability of disordered proteins to cell-cycle stage, environmental influences or tissue type.

Evidence for fuzziness has been curated by the Fuxreiter group since 2015 [Miskei et al. 2017] in the FuzDB database and recently been used to develop a prediction algorithm [unpublished], that according to Professor Fuxreiter achieves highly accurate predictions of fuzziness on a comprehensive validation dataset.

Both the implications of fuzziness for the understanding of the mode of action for disordered proteins (and disordered regions in otherwise ordered proteins) certainly spiked our interest, not least due to the potential importance of a clear understanding of these mode of actions for drug development.

Investigation of mutually exclusive splicing events using the CATH FunFam framework

The last of the 4 talks we would like to single out in this blogpost highlighted recent progress in using structure-based databases for the investigation of complex cellular events.

Christine Orengo from UCL presented her group’s work on mutually exclusive splicing, which employed the FunFam framework of the CATH database to probe the structural and functional implications of these splicing events [Lam et al. (2018), under review].

The FunFams are a subcategory of CATH’s homologous superfamilies, which further divides the superfamilies based on clusters of residue conservation within each family, thus creating groupings of functionally related proteins [Rentzsch & Orengo (2013)].

Mutually exclusive splicing that were investigated using this framework are a group of splicing events in which only one of several specific exons is present in the spliced mRNA. These exons usually show a high level of sequence similarity, leading to a low disruption of the protein structure by the splicing event. It is thought that this feature is a reason for the relative enrichment of mutually exclusive exons amongst alternative splicing events in the proteome.

This high degree of sequence similarity further enabled the mapping of the mutually exclusive exons to FunFams in the CATH database and thus further onto protein structures. This allowed the Orengo group to conduct a ‘large scale systematic study of the structural/functional effects of MXE splicing’.

Their analysis found that variable residues between the exons are significantly enriched at the protein surface, both compared to other stretches of the protein sequence and compared to non-variable residues in the exons, and in close proximity (< 6 Angstroms) to functional sites of the protein.

The main conclusion drawn from these findings was that, as previously hypothesised, mutually exclusive exons are likely functional switches, since changes in the surface exposed area close to functional sites are likely to affect the protein function without strongly disrupting its structure.

In the eyes of the Orengo group, this makes these splicing events good candidates for drug targeting, particularly in cases where a tissue specific isoform can be drugged, since in that case off-target effects could potentially be significantly reduced.

Sources:

Fujishima et al. (2018). Reconstruction of cysteine biosynthesis using engineered cysteine-free enzymes. Scientific Reports

Hlevnjak & Zagrovic (2015). Malleable nature of mRNA-protein compositional complementarity and its functional significance. Nucleic Acids Res

Lam, S. D., Orengo, C., & Lees, J. (2018). Protein structure and function analyses to understand the implication of mutually exclusive splicing. BioRxiv

Miskei, M. et al (2017). FuzDB: Database of fuzzy complexes, a tool to develop stochastic structure-function relationships for protein complexes and higher-order assemblies. Nucleic Acids Research

Polyansky & Zagrovic (2013). Evidence of direct complementary interactions between messenger RNAs and their cognate proteins. Nucleic Acids Res

Rentzsch, R., & Orengo, C. A. (2013). Protein function prediction using domain families. BMC Bioinformatics

Tompa, P., & Fuxreiter, M. (2008). Fuzzy complexes: polymorphism and structural disorder in protein-protein interactions. Trends in Biochemical Sciences