High-Throughput Reconstruction of Ancestral Protein Sequence, Structure, and Molecular Function

Aadland, Kelsey; Pugh, Charles; Kolaczkowski, Bryan

doi:10.1007/978-1-4939-8736-8_8

Cited by 21 publications

(21 citation statements)

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“…To assess ancestral sequence robustness to phylogenetic uncertainty (Hanson‐Smith, Kolaczkowski, & Thornton, ), ancestors inferred from the top five phylogenies ranked by log‐likelihood scores were selected for further analysis (Table , Appendix S2). Finally, to evaluate the effects of ambiguously reconstructed sites on subsequent structural analyses, Bayesian‐sampled ancestors were inferred from the maximum‐likelihood site posterior probabilities calculated by CODEML (Aadland et al, ). One hundred random Bayesian sequences were generated for each of the five ancestral nodes of interest across the top five phylogenies.…”

Section: Methodsmentioning

confidence: 99%

“…Here, we explored the indicators of nitrogenase metal usage history by a combined method relying on ancestral sequence reconstruction, an evolutionary approach by which inferred, historical protein sequence information can be linked to functional inferences of molecular properties evidenced by computational analyses or laboratory experiments (Aadland, Pugh, & Kolaczkowski, ; Benner, Sassi, & Gaucher, ; Thornton, ). These paleogenetic approaches have been increasingly applied in biogeochemically relevant molecular studies to offer insights into the coevolution of life and Earth (Garcia & Kacar, ; Gomez‐Fernandez et al, ; Kacar, Hanson‐Smith, Adam, & Boekelheide, ).…”

Section: Introductionmentioning

confidence: 99%

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Reconstructing the evolutionary history of nitrogenases: Evidence for ancestral molybdenum‐cofactor utilization

et al. 2020

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The nitrogenase metalloenzyme family, essential for supplying fixed nitrogen to the biosphere, is one of life's key biogeochemical innovations. The three forms of nitrogenase differ in their metal dependence, each binding either a FeMo-, FeV-, or FeFecofactor where the reduction of dinitrogen takes place. The history of nitrogenase metal dependence has been of particular interest due to the possible implication that ancient marine metal availabilities have significantly constrained nitrogenase evolution over geologic time. Here, we reconstructed the evolutionary history of nitrogenases, and combined phylogenetic reconstruction, ancestral sequence inference, and structural homology modeling to evaluate the potential metal dependence of ancient nitrogenases. We find that active-site sequence features can reliably distinguish extant Mo-nitrogenases from V-and Fe-nitrogenases and that inferred ancestral sequences at the deepest nodes of the phylogeny suggest these ancient proteins most resemble modern Mo-nitrogenases. Taxa representing early-branching nitrogenase lineages lack one or more biosynthetic nifE and nifN genes that both contribute to the assembly of the FeMo-cofactor in studied organisms, suggesting that early Monitrogenases may have utilized an alternate and/or simplified pathway for cofactor biosynthesis. Our results underscore the profound impacts that protein-level innovations likely had on shaping global biogeochemical cycles throughout the Precambrian, in contrast to organism-level innovations that characterize the Phanerozoic Eon. K E Y W O R D Sancestral sequence reconstruction, metal cofactor, metalloenzyme, nitrogen fixation, nitrogenase This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

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Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Reconstructing the evolutionary history of nitrogenases: Evidence for ancestral molybdenum‐cofactor utilization

et al. 2020

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“…To assess the effect of the template cofactor type on the 237 generated structure, additional models were constructed by specifying the inclusion of the FeV-238 cofactor of the 56NY VnfD template (D. Sippel & Einsle, 2017) (Appendix S5). 100 modeling 239 replicates were performed per sequence and assessed by averaging over the scaled Modeller 240 objective function, Discrete Optimized Protein Energy, and high resolution Discrete Optimized 241 Protein Energy scores, as previously described (Aadland et al, 2019). The ten best modeling 242 replicates per extant sequence, ten best replicates per maximum likelihood ancestral sequence, and 243 the single best replicate per Bayesian-sampled variant sequence were selected for further analysis, 244 totaling 3,080 models with the FeMo-cofactor specified.…”

Section: Structural Homology Modeling Of Extant and Ancestral Nitrogementioning

confidence: 99%

“…Thus, the evolutionary trajectory of nitrogenase metal 141 usage -and by extension the link between nitrogenase evolution and marine metal availabilities 142 over geologic time -is not yet known. 143 144 Here, we explored the indicators of nitrogenase metal usage history by a combinatorial method 145 relying on ancestral sequence reconstruction, a method by which inferred, historical protein 146 sequence information can be linked to functional molecular properties evidenced by computed 147 structures or laboratory experiments (Aadland, Pugh, & Kolaczkowski, 2019;Benner, Sassi, & 148 Gaucher, 2007;Thornton, 2004). These paleogenetic approaches have been increasingly applied 149 in biogeochemically relevant molecular studies to offer insights into the coevolution of life and 150…”

mentioning

confidence: 99%

“…To assess 207 ancestral sequence robustness to phylogenetic uncertainty (Hanson-Smith, Kolaczkowski, & 208 Thornton, 2010), ancestors inferred from the top five phylogenies ranked by log likelihood scores 209 were selected for further analysis (Table 1, Appendix S2). Finally, to evaluate the effects of 210 ambiguously reconstructed sites on subsequent structural analyses, Bayesian sampled ancestors 211 were inferred from the maximum likelihood site posterior probabilities calculated by CODEML 212 (Aadland et al, 2019). 100 random Bayesian sequence were generated for each of five ancestral 213 nodes of interest across the top five phylogenies.…”

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Reconstructing the Evolutionary History of Nitrogenases: Evidence for Ancestral Molybdenum-Cofactor Utilization

Garcia

McShea

Kolaczkowski

et al. 2019

Preprint

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15The nitrogenase metalloenzyme family, essential for supplying fixed nitrogen to the biosphere, is 16 one of life's key biogeochemical innovations. The three isozymes of nitrogenase differ in their 17 metal dependence, each binding either a FeMo-, FeV-, or FeFe-cofactor for the reduction of 18 nitrogen. The history of nitrogenase metal dependence has been of particular interest due to the 19 possible implication that ancient marine metal availabilities have significantly constrained 20 nitrogenase evolution over geologic time. Here, we combine phylogenetics and ancestral sequence 21 reconstruction -a method by which inferred, historical protein sequence information can be 22 linked to functional molecular properties -to reconstruct the metal dependence of ancient 23 nitrogenases. Inferred ancestral nitrogenase sequences at the deepest nodes of the phylogeny 24

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Structure and Activity of Reconstructed Pseudo‐Ancestral Cyclotides

Jiang,

Huang,

Kaas

et al. 2024

ChemMedChem

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Cyclotides are cyclic peptides that are promising scaffolds for the design of drug candidates and chemical tools. However, despite there being hundreds of reported cyclotides, drug design studies have commonly focussed on a select few prototypic examples. Here, we explored whether ancestral sequence reconstruction could be used to generate new cyclotides for further optimization. We show that the reconstructed ‘pseudo‐ancestral’ sequences, named Ancy‐m (for the ancestral cyclotide of the Möbius sub‐family) and Ancy‐b (for the bracelet sub‐family), have well‐defined structures like their extant members, comprising the core structural feature of a cyclic cystine knot. This motif underpins efforts to re‐engineer cyclotides for agrochemical and therapeutic applications. We further show that the reconstructed sequences are resistant to temperatures approaching boiling, bind to phosphatidyl‐ethanolamine lipid bilayers at micromolar affinity, and inhibit the growth of insect cells at inhibitory concentrations in the micromolar range. Interestingly, the Ancy‐b cyclotide had a higher oxidative folding yield than its comparator cyclotide cyO2, which belongs to the bracelet cyclotide subfamily known to be notoriously difficult to fold. Overall, this study provides new cyclotide sequences not yet found naturally that could be valuable starting points for the understanding of cyclotide evolution and for further optimization as drug leads.

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High-Throughput Reconstruction of Ancestral Protein Sequence, Structure, and Molecular Function

Cited by 21 publications

References 100 publications

Reconstructing the evolutionary history of nitrogenases: Evidence for ancestral molybdenum‐cofactor utilization

Reconstructing the evolutionary history of nitrogenases: Evidence for ancestral molybdenum‐cofactor utilization

Reconstructing the Evolutionary History of Nitrogenases: Evidence for Ancestral Molybdenum-Cofactor Utilization

Structure and Activity of Reconstructed Pseudo‐Ancestral Cyclotides

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