What Makes a Protein Fold Amenable to Functional Innovation? Fold Polarity and Stability Trade-offs

Dellus-Gur, Eynat; Tóth-Petróczy, Ágnes; Elias, Mikael; Tawfik, Dan S.

doi:10.1016/j.jmb.2013.03.033

Cited by 144 publications

(152 citation statements)

References 55 publications

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“…The directed evolution and bioinformatics analysis of a lactamase supports the model that enzymes are more capable of catalyzing multiple reactions when the active site is composed of loops juxtaposed to, but separate from, the core scaffold (43). Domain insertion can be considered an extreme example of this observation.…”

Section: Discussionmentioning

confidence: 87%

Panoramic view of a superfamily of phosphatases through substrate profiling

Huang

Pandya

Liu

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

132

139

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Large-scale activity profiling of enzyme superfamilies provides information about cellular functions as well as the intrinsic binding capabilities of conserved folds. Herein, the functional space of the ubiquitous haloalkanoate dehalogenase superfamily (HADSF) was revealed by screening a customized substrate library against >200 enzymes from representative prokaryotic species, enabling inferred annotation of ∼35% of the HADSF. An extremely high level of substrate ambiguity was revealed, with the majority of HADSF enzymes using more than five substrates. Substrate profiling allowed assignment of function to previously unannotated enzymes with known structure, uncovered potential new pathways, and identified isofunctional orthologs from evolutionarily distant taxonomic groups. Intriguingly, the HADSF subfamily having the least structural elaboration of the Rossmann fold catalytic domain was the most specific, consistent with the concept that domain insertions drive the evolution of new functions and that the broad specificity observed in HADSF may be a relic of this process.evolution | specificity | phosphatase | substrate screen | promiscuity S ince the first genomes were sequenced, there has been an exponential increase in the number of protein sequences deposited into databases worldwide. At the time of this writing the UniProtKB/TrEMBL database contains over 32 million protein sequences. Although this increase in sequence data has dramatically enhanced our understanding of the genomic organization of organisms, as the number of protein sequences grows, the proportion of firm functional assignments diminishes. Traditionally, methods of functional annotation involve comparing sequence identity between experimentally characterized proteins and newly sequenced ones, typically via BLAST (1). In cases where significant sequence similarity cannot be ascertained, proteins are annotated as "hypothetical" or "putative." Moreover, the decrease in sequence identity leads to an increased uncertainty in functional assignment, especially as the phylogenetic distance between organisms grows, limiting iso-functional ortholog discovery.As the number of newly sequenced genomes grows larger, more protein sequences are likely to be misannotated, oftentimes resulting in the propagation of incorrect functional annotation across newly identified sequences. To tackle the problem of unannotated or misannotated proteins, newer methods for computational assignment have been created with varying degrees of success (2). Although these methods outperform historical methods, continued improvement is necessary to ensure accurate annotation of function (2). A greater swath of functional space can be covered by screening substrates in a high-throughput manner on multiple enzymes from a family (3, 4). Family-wide substrate profiling offers a data-rich resource. The use of sparse screening of sequence space and a diversified library permits the determination of substrate specificity profiles to provide a familywide view of the range of substrates...

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Section: Discussionmentioning

confidence: 87%

Panoramic view of a superfamily of phosphatases through substrate profiling

Huang

Pandya

Liu

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

132

139

View full text Add to dashboard Cite

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“…DNA amplification was done with Taq DNA polymerase as described by Chao et al (41). The library was prepared based on a stabilized variant of the TEM1 β-lactamase previously described (42). The library size was estimated to be ∼10 8 by plating serial dilutions on selection plates lacking tryptophan.…”

Section: Methodsmentioning

confidence: 99%

Low-stringency selection of TEM1 for BLIP shows interface plasticity and selection for faster binders

Cohen-Khait

Schreiber

2016

Proc. Natl. Acad. Sci. U.S.A.

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Protein-protein interactions occur via well-defined interfaces on the protein surface. Whereas the location of homologous interfaces is conserved, their composition varies, suggesting that multiple solutions may support high-affinity binding. In this study, we examined the plasticity of the interface of TEM1 β-lactamase with its protein inhibitor BLIP by low-stringency selection of a random TEM1 library using yeast surface display. Our results show that most interfacial residues could be mutated without a loss in binding affinity, protein stability, or enzymatic activity, suggesting plasticity in the interface composition supporting high-affinity binding. Interestingly, many of the selected mutations promoted faster association. Further selection for faster binders was achieved by drastically decreasing the libraryligand incubation time to 30 s. Preequilibrium selection as suggested here is a novel methodology for specifically selecting faster-associating protein complexes.protein-protein interaction | in vitro evolution | association rate | interface plasticity P rotein-protein interactions play important roles in most cellular processes. Proteins interact through chemical and structural complementarity of their mutual binding sites (1). Amino acids found in physical proximity form noncovalent interactions that stabilize the complex (2). Residues not directly involved in binding also play a role in complex formation by stabilizing the association encounter complex and transition state, which determine the interaction's association rate constant (3). Protein interaction interfaces seem to have dual, seemingly contradicting, properties: On one hand, the interface is a unique area (4) on the protein surface that has evolved to bind specific partners (5). Although much is known about the molecular mechanism of binding, de novo design of protein interactions is still a complicated task, requiring in vitro evolution to achieve high-affinity binding (6-8). On the other hand, interfaces seem to be rather tolerant to mutagenesis (2, 9-11), implying that there are many ways to achieve high-affinity binding. The plastic nature of proteinprotein interfaces becomes particularly clear when comparing the conservation levels of transient complex interfaces versus surface and core residues in homologous complexes (12, 13). Here it is shown that core residues are similarly conserved as permanent interaction interface residues but that transient interaction interface residues are hardly more conserved than other surface residues. The relatively low conservation levels of transient interface residues also make it difficult to predict the protein binding sites (contrary to permanent interfaces) (14, 15). In fact, it was suggested that the vast majority of interfacial residues are important only for conformational optimization of the few interacting residues, resulting in variability in their actual identity (16).The interface formed between different β-lactamase proteins and their inhibitor protein BLIP is a good example of the use...

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“…[33,[138][139][140] As loop regions are generally not tethered to the enzyme by extensive intra-protein amino acid interactions, they can adopt a greater range of conformations and mutations are less likely to perturb the protein fold. [141] There is often an accumulation of mutations in the loop regions in xenobiotic-degrading enzymes correlated with the new function. This has been observed extensively in the natural, designed, and directed evolution towards enhanced organophosphate hydrolysis in proteins from several folds.…”

Section: Loop Rearrangements and Flexibility Promote Novel Functionmentioning

confidence: 99%

The Evolution of New Catalytic Mechanisms for Xenobiotic Hydrolysis in Bacterial Metalloenzymes

et al. 2016

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An increasing number of bacterial metalloenzymes have been shown to catalyse the breakdown of xenobiotics in the environment, while others exhibit a variety of promiscuous xenobiotic-degrading activities. Several different evolutionary processes have allowed these enzymes to gain or enhance xenobiotic-degrading activity. In this review, we have surveyed the range of xenobiotic-degrading metalloenzymes, and discuss the molecular and catalytic basis for the development of new activities. We also highlight how our increased understanding of the natural evolution of xenobiotic-degrading metalloenzymes can be been applied to laboratory enzyme design.

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What Makes a Protein Fold Amenable to Functional Innovation? Fold Polarity and Stability Trade-offs

Cited by 144 publications

References 55 publications

Panoramic view of a superfamily of phosphatases through substrate profiling

Panoramic view of a superfamily of phosphatases through substrate profiling

Low-stringency selection of TEM1 for BLIP shows interface plasticity and selection for faster binders

The Evolution of New Catalytic Mechanisms for Xenobiotic Hydrolysis in Bacterial Metalloenzymes

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