Recent News

An Evolutionary History of the CoA-Binding Protein Nat/Ivy [ Link ]
Liam M. Longo*, Hayate Hirai, Shawn E. MyGlynn*
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Nat/Ivy is a diverse and ubiquitous CoA-binding evolutionary lineage that catalyzes acyltransferase reactions, primarily converting thioesters into amides. At the heart of the Nat/Ivy fold is a phosphate-binding loop that bears a striking resemblance to that of P-loop NTPases – both are extended, glycine-rich loops situated between a β-strand and an α-helix. Nat/Ivy, therefore, represents an intriguing intersection between thioester chemistry, a putative primitive energy currency, and an ancient mode of phospho-ligand binding. Current evidence suggests that Nat/Ivy emerged independently of other cofactor-utilizing enzymes, and that the observed structural similarity – particularly of the cofactor binding site – is the product of shared constraints instead of shared ancestry. The reliance of Nat/Ivy on a β-α-β motif for CoA binding highlights the extent to which this simple structural motif may have been a fundamental evolutionary ‘nucleus’ around which modern cofactor-binding domains condensed, as has been suggested for HUP domains, Rossmanns, and P-loop NTPases. Finally, by dissecting the patterns of conserved interactions between Nat/Ivy families and CoA, the coevolution of the enzyme and the cofactor was analyzed. As with the Rossmann, it appears that the pyrophosphate moiety at the center of the cofactor predates the enzyme, suggesting that Nat/Ivy emerged sometime after the metabolite dephospho-CoA.

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Stunning views and exciting talks at the Annual Meeting of the Biophysical Society of Japan. Check out my poster, describing a project in collaboration with the Prof. Norman Metanis and Prof. Koby Levy, here!

On the Continuity Between Ancient Geochemistry and Modern Biochemistry
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Although OIST is surrounded by stunning beaches, the real draw is the cutting-edge research happening on campus. Thanks so much to fellow Tawfik Lab alumnus Prof. Paola Laurino for inviting me!

Utilization of Diverse Organophosphorus Pollutants by Marine Bacteria [ Link ]
Dragana Despotović*, Einav Aharon^, Olena Trofimyuk^, Artem Dubovetskyi^, Kesava Cherukuri^, Yacov Ashani, Or Eliason, Martin Sperfeld, Haim Leader, Andrea Castelli, Laura Fumagalli, Alon Savidor, Yishai Levin, Liam M. Longo*, Einat Segev*, Dan S. Tawfik
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Anthropogenic organophosphorus compounds (AOPCs), such as phosphotriesters, are used extensively as plasticizers, flame retardants, nerve agents, and pesticides. To date, only a handful of soil bacteria bearing a phosphotriesterase (PTE), the key enzyme in the AOPC degradation pathway, have been identified. Therefore, the extent to which bacteria are capable of utilizing AOPCs as a phosphorus source, and how widespread this adaptation may be, remains unclear. Marine environments with phosphorus limitation and increasing levels of pollution by AOPCs may drive the emergence of PTE activity. Here, we report the utilization of diverse AOPCs by four model marine bacteria and 17 bacterial isolates from the Mediterranean Sea and the Red Sea. To unravel the details of AOPC utilization, two PTEs from marine bacteria were isolated and characterized, with one of the enzymes belonging to a protein family that, to our knowledge, has never before been associated with PTE activity. When expressed in Escherichia coli with a phosphodiesterase, a PTE isolated from a marine bacterium enabled growth on a pesticide analog as the sole phosphorus source. Utilization of AOPCs may provide bacteria a source of phosphorus in depleted environments and offers a prospect for the bioremediation of a pervasive class of anthropogenic pollutants.

Peptide-RNA Coacervates as a Cradle for the Evolution of Folded Domains [ Link ]
Manas Seal, Orit Weil-Ktorza, Dragana Despotović, Dan S. Tawfik, Yaakov Levy, Norman Metanis, Liam M. Longo*, and Daniella Goldfarb*
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Peptide-RNA coacervates can result in the concentration and compartmentalization of simple biopolymers. Given their primordial relevance, peptide-RNA coacervates may have also been a key site of early protein evolution. However, the extent to which such coacervates might promote or suppress the exploration of novel peptide conformations is fundamentally unknown. To this end, we used electron paramagnetic resonance spectroscopy (EPR) to characterize the structure and dynamics of an ancient and ubiquitous nucleic acid binding element, the helix-hairpin-helix (HhH) motif, alone and in the presence of RNA, with which it forms coacervates. Double electron–electron resonance (DEER) spectroscopy applied to singly labeled peptides containing one HhH motif revealed the presence of dimers, even in the absence of RNA. Moreover, dimer formation is promoted upon RNA binding and was detectable within peptide-RNA coacervates. DEER measurements of spin-diluted, doubly labeled peptides in solution indicated transient α-helical character. The distance distributions between spin labels in the dimer and the signatures of α-helical folding are consistent with the symmetric (HhH)2-Fold, which is generated upon duplication and fusion of a single HhH motif and traditionally associated with dsDNA binding. These results support the hypothesis that coacervates are a unique testing ground for peptide oligomerization and that phase-separating peptides could have been a resource for the construction of complex protein structures via common evolutionary processes, such as duplication and fusion.

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The Blue Marble Space Institute of Science (BMSIS) is an international group of scientists interested in basic science, sustainability, and public engagement. I can't wait to start learning together with the kindred sprints at BMSIS!

IdeasLab: Bringing Chemistry, Physics and Computing to Life
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At the IdeasLab, I had the opportunity to work alongside computer scientists and physicists as we developed projects to probe the nature of mind, agency, and the process of abiogenesis. Many thanks to the John Templeton Foundation for making this trip possible -- I learned so much from my peers and mentors, and it's a week I won't soon forget!

Check out made-for-YouTube versions of the talks here. As these papers are not yet published, the stories are subject to change! A collaboration between my lab and the Metanis Lab (Hebrew University of Jerusalem) about "ambidextrous" protein folds: A peek at some conversations between me and my former Postdoctoral Advisor Shawn McGlynn:

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I've been promoted to Specially Appointed Associate Professor at the Earth-Life Science Institute (ELSI) at the Tokyo Institute of Technology! If you are interested protein evolution or metabolism and would like to join the Longo Lab @ ELSI (Master's or PhD level), please drop me a line!

Evidence for the Emergence of β-Trefoils by ‘Peptide Budding’ from an IgG-like β-Sandwich [ Link ]
Liam M. Longo*, Rachel Kolodny*, Shawn E. McGlynn*
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As sequence and structure comparison algorithms gain sensitivity, the intrinsic interconnectedness of the protein universe has become increasingly apparent. Despite this general trend, β-trefoils have emerged as an uncommon counterexample: They are an isolated protein lineage for which few, if any, sequence or structure associations to other lineages have been identified. If β-trefoils are, in fact, remote islands in sequence-structure space, it implies that the oligomerizing peptide that founded the β-trefoil lineage itself arose de novo. To better understand β-trefoil evolution, and to probe the limits of fragment sharing across the protein universe, we identified both ‘β-trefoil bridging themes’ (evolutionarily-related sequence segments) and ‘β-trefoil-like motifs’ (structure motifs with a hallmark feature of the β-trefoil architecture) in multiple, ostensibly unrelated, protein lineages. The success of the present approach stems, in part, from considering β-trefoil sequence segments or structure motifs rather than the β-trefoil architecture as a whole, as has been done previously. The newly uncovered inter-lineage connections presented here suggest a novel hypothesis about the origins of the β-trefoil fold itself – namely, that it is a derived fold formed by ‘budding’ from an Immunoglobulin-like β-sandwich protein. These results demonstrate how the evolution of a folded domain from a peptide need not be a signature of antiquity and underpin an emerging truth: few protein lineages escape nature’s sewing table.

Dan Salah Tawfik (1955 - 2021): Pioneer of Molecular Evolution [ Link ]
Liam M. Longo, Dragana Despotović, and Lianet Noda-García
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We are deeply saddened by the premature and unexpected passing of our dear friend and mentor Dan Salah Tawfik (דן סלח תופיק). Danny’s contributions to science were immense, and his insights into enzyme catalysis have shaped an array of fields. In this obituary, we try to communicate the impact Danny had on us personally as a mentor, and the joy of doing science in Danny's lab. Photo by David Salem of Zoog Productions.

The evolutionary history of the HUP domain [ Link ]
Ita Gruic-Sovulj*, Liam M. Longo, Jagoda Jabłońska, and Dan S. Tawfik*
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Among the enzyme lineages that undoubtedly emerged prior to the last universal common ancestor is the so-called HUP, which includes Class I aminoacyl tRNA synthetases (AARSs) as well as enzymes mediating NAD, FAD, and CoA biosynthesis. Here, we provide a detailed analysis of HUP evolution, from emergence to structural and functional diversification. The HUP is a nucleotide binding domain that uniquely catalyzes adenylation via the release of pyrophosphate. In contrast to other ancient nucleotide binding domains with the aba sandwich architecture, such as P-loop NTPases, the HUP’s most conserved feature is not phosphate binding, but rather ribose binding by backbone interactions to the tips of b1 and/or b4. Indeed, the HUP exhibits unusual evolutionary plasticity and, while ribose binding is conserved, the location and mode of binding to the base and phosphate moieties of the nucleotide, and to the substrate(s) reacting with it, have diverged with time, foremost along the emergence of the AARSs. The HUP also beautifully demonstrates how a well-packed scaffold com- bined with evolvable surface elements promotes evolutionary innovation. Finally, we offer a scenario for the emergence of the HUP from a seed bab fragment, and suggest that despite an identical architecture, the HUP and the Rossmann represent independent emergences.

Helicase-Like Functions in Phosphate Loop Containing Beta-Alpha Polypeptides [ Link ]
Pratik Vyas, Olena Trofimyuk, Liam M. Longo, Fanindra Kumar Deshmukh, Michal Sharon, and Dan S. Tawfik*
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It is widely assumed that today’s large and complex proteins emerged from much shorter and simpler polypeptides. Yet the nature of these early precursors remains enigmatic. We describe polypeptides that contain one of the earliest protein motifs, a phosphate-binding loop, or P-loop, embedded in a single beta-alpha element. These P-loop prototypes show intriguing characteristics of a primordial world comprised of nucleic acids and peptides. They are ‘generalists’ capable of binding different phospho-ligands, including inorganic polyphosphates and single-stranded DNA. Nonetheless, in promoting double-stranded DNA unwinding and strand-exchange they resemble modern P-loop helicases and recombinases. Our study describes a missing link in the evolution of complex proteins – simple polypeptides that tangibly relate to contemporary P-loop enzymes in sequence, structure and function.

On the Emergence of P-Loop NTPase and Rossmann Enzymes from a Beta-Alpha-Beta Ancestral Fragment [ Link ]
Liam M. Longo, Jagoda Jabłońska, Pratik Vyas, Manil Kanade, Rachel Kolodny*, Nir Ben-Tal*, and Dan S. Tawfik*
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Dating back to the last universal common ancestor (LUCA), the P-loop NTPases and Rossmanns now comprise the most ubiquitous and diverse enzyme lineages. Intriguing similarities in their overall architecture and phosphate binding motifs suggest common ancestry; however, due to a lack of sequence identity and some fundamental structural differences, these families are considered independent emergences. To address this longstanding dichotomy, we systematically searched for ‘bridge proteins’ with structure and sequence elements shared by both lineages. We detected homologous segments that span the first βαβ segment of both lineages and include two key functional motifs: (i) a phosphate binding loop – the ‘Walker A’ motif of P-loop NTPases or the Rossmann equivalent, both residing at the N-terminus of α1; and (ii) an Asp at the tip of β2. The latter comprises the ‘Walker B’ aspartate that chelates the catalytic metal in P-loop NTPases, or the canonical Rossmann β2-Asp that binds the cofactor’s ribose moiety. Tubulin, a Rossmann GTPase, demonstrates the potential of the β2-Asp to take either one of these two roles. We conclude that common P-loops/Rossmann ancestry is plausible, although convergence cannot be completely ruled out. Regardless, both lineages most likely emerged from a polypeptide comprising a βαβ segment carrying the above two functional motifs, a segment that comprises the core of both enzyme families to this very day.

Polyamines Mediate Folding of Primordial Hyperacidic Helical Proteins [ Link ]
Dragana Despotović*^, Liam M. Longo^, Einav Aharon, Amit Kahana, Tali Scherf, Ita Gruic-Sovulj, and Dan S. Tawfik*
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Polyamines are known to mediate diverse biological processes, and specifically to bind and stabilize compact conformations of nucleic acids, acting as chemical chaperones that promote folding by offsetting the repulsive negative charges of the phosphodiester backbone. However, whether and how polyamines modulate the structure and function of proteins remains unclear. Further, early proteins are thought to have been highly acidic, like nucleic acids, due to a scarcity of basic amino acids in the prebiotic context. Perhaps polyamines, the abiotic synthesis of which is simple, could have served as chemical chaperones for such primordial proteins? We replaced all lysines of an ancestral 60-residue helix-bundle protein to glutamate, resulting in a disordered protein with 21 glutamates in total. Polyamines efficiently induce folding of this hyper-acidic protein at sub-millimolar concentrations, and their potency scaled with the number of amine groups. Compared to cations, polyamines were several orders of magnitude more potent than Na+, while Mg2+ and Ca2+ had an effect similar to a di-amine, inducing folding at approximately seawater concentrations. We propose that (i) polyamines and dications may have had a role in promoting folding of early proteins devoid of basic residues, and that (ii) coil-helix transitions could be the basis of polyamine regulation in contemporary proteins.

Several articles were written about Primordial emergence of a nucleic acid-binding protein via phase separation and statistical ornithine-to-arginine conversion, including:
Primordial emergence of a nucleic acid-binding protein via phase separation and statistical ornithine-to-arginine conversion [ Link ]
Liam M. Longo^, Dragana Despotović^, Orit Weil-Ktorza^, Matthew J. Walker, Jagoda Jabłońska, Yael Fridmann-Sirkis, Gabriele Varani, Norman Metanis*, and Dan S. Tawfik*
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The first proteins emerged some 4 billion years ago, and understanding how they came about is a daunting challenge. Further complicating matters, the rules of protein structure and function derived from modern proteins may be irrelevant to their earliest ancestors. We report an integrated approach in which protein sequence, structure, and function are considered. We show that a simple function (phase separation) may have served as the basis for a complex function (specific double-stranded DNA binding), and that disordered polypeptides can give rise to structured, well-packed domains. Finally, we demonstrate that functional proteins may arise from short and simple sequences that include ornithine, an amino acid likely present in early proteins yet absent in modern proteins.

Ab initio folding of a trefoil‐fold motif reveals structural similarity with a β‐propeller blade motif [ Link ]
Connie A. Tenorio, Liam M. Longo, Joseph B. Parker, Jihun Lee, and Michael Blaber*
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Many protein architectures exhibit evidence of internal rotational symmetry postulated to be the result of gene duplication/fusion events involving a primordial polypeptide motif. A common feature of such structures is a domain-swapped arrangement at the interface of the N- and C-termini motifs and postulated to provide cooperative interactions that promote folding and stability. De novo designed symmetric protein architectures have demonstrated an ability to accommodate circular permutation of the N- and C-termini in the overall architecture; however, the folding requirement of the primordial motif is poorly understood, and tolerance to circular permutation is essentially unknown. The β-trefoil protein fold is a threefold-symmetric architecture where the repeating ~42-mer "trefoil-fold" motif assembles via a domain-swapped arrangement. The trefoil-fold structure in isolation exposes considerable hydrophobic area that is otherwise buried in the intact β-trefoil trimeric assembly. The trefoil-fold sequence is not predicted to adopt the trefoil-fold architecture in ab initio folding studies; rather, the predicted fold is closely related to a compact "blade" motif from the β-propeller architecture. Expression of a trefoil-fold sequence and circular permutants shows that only the wild-type N-terminal motif definition yields an intact β-trefoil trimeric assembly, while permutants yield monomers. The results elucidate the folding requirements of the primordial trefoil-fold motif, and also suggest that this motif may sample a compact conformation that limits hydrophobic residue exposure, contains key trefoil-fold structural features, but is more structurally homologous to a β-propeller blade motif.

Short and simple sequences favored the emergence of N-helix phospho-ligand binding sites in the first enzymes. [ Link ]
Liam M. Longo, Dušan Petrović, Shina Caroline Lynn Kamerlin, and Dan S. Tawfik*
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The first enzymes emerged ∼4 billion years ago and have subsequently become the most diverse and functionally important component of life. But what were the first enzymes doing and how did they look? We probed the properties of the first enzymes by analyzing phospho-ligand binding across all known protein evolutionary lineages. We find that phospho-ligand binding was the founding function of the most ancient enzymes. As opposed to younger evolutionary lineages, ancient enzymes preferentially use N termini of α-helices to bind phosphate moieties. The dominance of N-helix binding sites in the earliest enzymes reflects the ability of the α-helix to realize binding via short and simple sequences, including serines and threonines that interact via both the backbone and side chain.