OpenFold: retraining AlphaFold2 yields new insights into its learning mechanisms and capacity for generalization.

AlphaFold2 revolutionized structural biology with the ability to predict protein structures with exceptionally high accuracy. Its implementation, however, lacks the code and data required to train new models. These are necessary to (1) tackle new tasks, like protein-ligand complex structure prediction, (2) investigate the process by which the model learns and (3) assess the model's capacity to generalize to unseen regions of fold space. Here we report OpenFold, a fast, memory efficient and trainable implementation of AlphaFold2. We train OpenFold from scratch, matching the accuracy of AlphaFold2. Having established parity, we find that OpenFold is remarkably robust at generalizing even when the size and diversity of its training set is deliberately limited, including near-complete elisions of classes of secondary structure elements. By analyzing intermediate structures produced during training, we also gain insights into the hierarchical manner in which OpenFold learns to fold. In sum, our studies demonstrate the power and utility of OpenFold, which we believe will prove to be a crucial resource for the protein modeling community.

Computational design of a red fluorophore ligase for site-specific protein labeling in living cells.

Chemical fluorophores offer tremendous size and photophysical advantages over fluorescent proteins but are much more challenging to target to specific cellular proteins. Here, we used Rosetta-based computation to design a fluorophore ligase that accepts the red dye resorufin, starting from Escherichia coli lipoic acid ligase. X-ray crystallography showed that the design closely matched the experimental structure. Resorufin ligase catalyzed the site-specific and covalent attachment of resorufin to various cellular proteins genetically fused to a 13-aa recognition peptide in multiple mammalian cell lines and in primary cultured neurons. We used resorufin ligase to perform superresolution imaging of the intermediate filament protein vimentin by stimulated emission depletion and electron microscopies. This work illustrates the power of Rosetta for major redesign of enzyme specificity and introduces a tool for minimally invasive, highly specific imaging of cellular proteins by both conventional and superresolution microscopies.

Automating human intuition for protein design.

In the design of new enzymes and binding proteins, human intuition is often used to modify computationally designed amino acid sequences prior to experimental characterization. The manual sequence changes involve both reversions of amino acid mutations back to the identity present in the parent scaffold and the introduction of residues making additional interactions with the binding partner or backing up first shell interactions. Automation of this manual sequence refinement process would allow more systematic evaluation and considerably reduce the amount of human designer effort involved. Here we introduce a benchmark for evaluating the ability of automated methods to recapitulate the sequence changes made to computer-generated models by human designers, and use it to assess alternative computational methods. We find the best performance for a greedy one-position-at-a-time optimization protocol that utilizes metrics (such as shape complementarity) and local refinement methods too computationally expensive for global Monte Carlo (MC) sequence optimization. This protocol should be broadly useful for improving the stability and function of designed binding proteins.

De novo design of a non-local ß-sheet protein with high stability and accuracy.

ß-sheet proteins carry out critical functions in biology, and hence are attractive scaffolds for computational protein design. Despite this potential, de novo design of all-ß-sheet proteins from first principles lags far behind the design of all-α or mixed-αß domains owing to their non-local nature and the tendency of exposed ß-strand edges to aggregate. Through study of loops connecting unpaired ß-strands (ß-arches), we have identified a series of structural relationships between loop geometry, side chain directionality and ß-strand length that arise from hydrogen bonding and packing constraints on regular ß-sheet structures. We use these rules to de novo design jellyroll structures with double-stranded ß-helices formed by eight antiparallel ß-strands. The nuclear magnetic resonance structure of a hyperthermostable design closely matched the computational model, demonstrating accurate control over the ß-sheet structure and loop geometry. Our results open the door to the design of a broad range of non-local ß-sheet protein structures.

Two distinct binding modes of a protein cofactor with its target RNA.

Like most cellular RNA enzymes, the bI5 group I intron requires binding by a protein cofactor to fold correctly. Here, we use single-molecule approaches to monitor the structural dynamics of the bI5 RNA in real time as it assembles with its CBP2 protein cofactor. These experiments show that CBP2 binds to the target RNA in two distinct modes with apparently opposite effects: a "non-specific" mode that forms rapidly and induces large conformational fluctuations in the RNA, and a "specific" mode that forms slowly and stabilizes the native RNA structure. The bI5 RNA folds though multiple pathways toward the native state, typically traversing dynamic intermediate states induced by non-specific binding of CBP2. These results suggest that the protein cofactor-assisted RNA folding involves sequential non-specific and specific protein-RNA interactions. The non-specific interaction potentially increases the local concentration of CBP2 and the number of conformational states accessible to the RNA, which may promote the formation of specific RNA-protein interactions.

All-atom Monte Carlo simulation of GCAA RNA folding.

We report a detailed all-atom simulation of the folding of the GCAA RNA tetraloop. The GCAA tetraloop motif is a very common and thermodynamically stable secondary structure in natural RNAs. We use our simulation methods to study the folding behavior of a 12-base GCAA tetraloop structure with a four-base helix adjacent to the tetraloop proper. We implement an all-atom Monte Carlo (MC) simulation of RNA structural dynamics using a Go potential. Molecular dynamics (MD) simulation of RNA and protein has realistic energetics and sterics, but is extremely expensive in terms of computational time. By coarsely treating non-covalent energetics, but retaining all-atom sterics and entropic effects, all-atom MC techniques are a useful method for the study of protein and now RNA. We observe a sharp folding transition for this structure, and in simulations at room temperature the state histogram shows three distinct minima: an unfolded state (U), a more narrow intermediated state (I), and a narrow folded state (F). The intermediate consists primarily of structures with the GCAA loop and some helix hydrogen bonds formed. Repeated kinetic folding simulations reveal that the number of helix base-pairs forms a simple 1D reaction coordinate for the I-->N transition.

Amino acid sequence of the Felis catus prion protein.

A new determination of the house cat (Felis catus) prion protein gene sequence (fPrnp), which has so far been subject of controversy, is reported. The newly determined fPrnp sequence is similar to dog (Canis familiaris) and mink (Mustela putorius) Prnp, but differs significantly from both fPrnp sequences that were previously deposited in the GenBank. Comparison of the canine and feline prion protein sequences suggests a set of amino acid replacements relative to bovine PrP that might relate to the observed different susceptibilities of the two species to TSE infection by ingestion of BSE-infected beef.

A Pareto-optimal refinement method for protein design scaffolds.

Computational design of protein function involves a search for amino acids with the lowest energy subject to a set of constraints specifying function. In many cases a set of natural protein backbone structures, or "scaffolds", are searched to find regions where functional sites (an enzyme active site, ligand binding pocket, protein-protein interaction region, etc.) can be placed, and the identities of the surrounding amino acids are optimized to satisfy functional constraints. Input native protein structures almost invariably have regions that score very poorly with the design force field, and any design based on these unmodified structures may result in mutations away from the native sequence solely as a result of the energetic strain. Because the input structure is already a stable protein, it is desirable to keep the total number of mutations to a minimum and to avoid mutations resulting from poorly-scoring input structures. Here we describe a protocol using cycles of minimization with combined backbone/sidechain restraints that is Pareto-optimal with respect to RMSD to the native structure and energetic strain reduction. The protocol should be broadly useful in the preparation of scaffold libraries for functional site design.

Computational design of enone-binding proteins with catalytic activity for the Morita-Baylis-Hillman reaction.

The Morita-Baylis-Hillman reaction forms a carbon-carbon bond between the α-carbon of a conjugated carbonyl compound and a carbon electrophile. The reaction mechanism involves Michael addition of a nucleophile catalyst at the carbonyl ß-carbon, followed by bond formation with the electrophile and catalyst disassociation to release the product. We used Rosetta to design 48 proteins containing active sites predicted to carry out this mechanism, of which two show catalytic activity by mass spectrometry (MS). Substrate labeling measured by MS and site-directed mutagenesis experiments show that the designed active-site residues are responsible for activity, although rate acceleration over background is modest. To characterize the designed proteins, we developed a fluorescence-based screen for intermediate formation in cell lysates, carried out microsecond molecular dynamics simulations, and solved X-ray crystal structures. These data indicate a partially formed active site and suggest several clear avenues for designing more active catalysts.

Thermodynamics and kinetics of the hairpin ribozyme from atomistic folding/unfolding simulations.

We report a set of atomistic folding/unfolding simulations for the hairpin ribozyme using a Monte Carlo algorithm. The hairpin ribozyme folds in solution and catalyzes self-cleavage or ligation via a specific two-domain structure. The minimal active ribozyme has been studied extensively, showing stabilization of the active structure by cations and dynamic motion of the active structure. Here, we introduce a simple model of tertiary-structure formation that leads to a phase diagram for the RNA as a function of temperature and tertiary-structure strength. We then employ this model to capture many folding/unfolding events and to examine the transition-state ensemble (TSE) of the RNA during folding to its active "docked" conformation. The TSE is compact but with few tertiary interactions formed, in agreement with single-molecule dynamics experiments. To compare with experimental kinetic parameters, we introduce a novel method to benchmark Monte Carlo kinetic parameters to docking/undocking rates collected over many single molecular trajectories. We find that topology alone, as encoded in a biased potential that discriminates between secondary and tertiary interactions, is sufficient to predict the thermodynamic behavior and kinetic folding pathway of the hairpin ribozyme. This method should be useful in predicting folding transition states for many natural or man-made RNA tertiary structures.

Prion protein NMR structures of cats, dogs, pigs, and sheep.

The NMR structures of the recombinant cellular form of the prion proteins (PrPC) of the cat (Felis catus), dog (Canis familiaris), and pig (Sus scrofa), and of two polymorphic forms of the prion protein from sheep (Ovis aries) are presented. In all of these species, PrPC consists of an N-terminal flexibly extended tail with approximately 100 amino acid residues and a C-terminal globular domain of approximately 100 residues with three alpha-helices and a short antiparallel beta-sheet. Although this global architecture coincides with the previously reported murine, Syrian hamster, bovine, and human PrPC structures, there are local differences between the globular domains of the different species. Because the five newly determined PrPC structures originate from species with widely different transmissible spongiform encephalopathy records, the present data indicate previously uncharacterized possible correlations between local features in PrPC three-dimensional structures and susceptibility of different mammalian species to transmissible spongiform encephalopathies.

Effects of chronic stress on hippocampal long-term potentiation.

Chronic stress causes atrophy of the apical dendrites of CA3 pyramidal neurons and deficits in spatial memory. We investigated the effects of chronic stress on hippocampal physiology and long-term potentiation (LTP) in the CA3 and dentate gyrus (DG). Rats were subjected to chronic (21 days, 6 h/day) restraint stress and tested for LTP 48 h following the last stress episode. Control animals were briefly handled each day, similar to the experimental group but without restraint. To eliminate acute stress effects, a second control group of rats was subjected to a single acute (6 h) restraint stress and tested for LTP 48 h later. Field potential recordings were made, under chloropent anesthesia, from the stratum lucidum of CA3, with stimulation of either the mossy fiber or commissural/associational pathways, or in the DG granule-cell layer, with stimulation of the medial perforant pathway. Chronic stress produced a suppression of LTP at 48 h compared to controls in a site-specific manner, namely, significantly lower LTP in the medial perforant input to the DG and also in the commissural/associational input to the CA3, but not in the mossy fiber input to CA3. The animals subjected to acute stress and tested 48 h later did not show a suppression in LTP. High-frequency stimulation (HFS) of the commissural/associational and mossy fiber inputs to CA3 produced epileptic afterdischarges in 56% of acutely stressed animals and in 29% of chronically stressed animals, whereas HFS caused afterdischarges in only 9% of nonstressed controls. No afterdischarges were seen in the medial perforant path input to DG. In order to explore the basis for these changes, we performed paired-pulse inhibition/facilitation (PPI/F) and current-source-density (CSD) analysis in stressed and control animals. For PPI/F, acute stress caused an overall significant enhancement of excitation in the commissural/associational input to CA3 and medial perforant path input to DG. In contrast, chronic stress did not produce significant changes in PPI/F. The CSD analysis revealed significant chronic stress-induced shifts in the current sources and sinks in the apical dendrites and pyramidal cell layers of the CA3 field but not in the DG. These results are consistent with the morphological findings for stress effects upon dendrites of CA3 neurons. Furthermore, they suggest that chronic stress produces changes in the input-output relationship in the hippocampal trisynaptic circuit which could affect information flow through this structure.