Bryan S. Der

Serverification of Molecular Modeling Applications: The Rosetta Online Server That Includes Everyone (ROSIE)

The Rosetta molecular modeling software package provides experimentally tested and rapidly evolving tools for the 3D structure prediction and high-resolution design of proteins, nucleic acids, and a growing number of non-natural polymers. Despite its free availability to academic users and improving documentation, use of Rosetta has largely remained confined to developers and their immediate collaborators due to the code’s difficulty of use, the requirement for large computational resources, and the unavailability of servers for most of the Rosetta applications. Here, we present a unified web framework for Rosetta applications called ROSIE (Rosetta Online Server that Includes Everyone). ROSIE provides (a) a common user interface for Rosetta protocols, (b) a stable application programming interface for developers to add additional protocols, (c) a flexible back-end to allow leveraging of computer cluster resources shared by RosettaCommons member institutions, and (d) centralized administration by the RosettaCommons to ensure continuous maintenance. This paper describes the ROSIE server infrastructure, a step-by-step ‘serverification’ protocol for use by Rosetta developers, and the deployment of the first nine ROSIE applications by six separate developer teams: Docking, RNA de novo, ERRASER, Antibody, Sequence Tolerance, Supercharge, Beta peptide design, NCBB design, and VIP redesign. As illustrated by the number and diversity of these applications, ROSIE offers a general and speedy paradigm for serverification of Rosetta applications that incurs negligible cost to developers and lowers barriers to Rosetta use for the broader biological community. ROSIE is available at http://rosie.rosettacommons.org.

Catalysis by a De Novo Zinc-Mediated Protein Interface: Implications for Natural Enzyme Evolution and Rational Enzyme Engineering

Here we show that a recent computationally designed zinc-mediated protein interface is serendipitously capable of catalyzing carboxyester and phosphoester hydrolysis. Although the original motivation was to design a de novo zinc-mediated protein-protein interaction (called MID1-zinc), we observed in the homodimer crystal structure a small cleft and open zinc coordination site. We investigated if the cleft and zinc site at the designed interface were sufficient for formation of a primitive active site that can perform hydrolysis. MID1-zinc hydrolyzes 4-nitrophenyl acetate with a rate acceleration of 10(5) and a k(cat)/K(M) of 630 M(-1) s(-1) and 4-nitrophenyl phosphate with a rate acceleration of 10(4) and a k(cat)/K(M) of 14 M(-1) s(-1). These rate accelerations by an unoptimized active site highlight the catalytic power of zinc and suggest that the clefts formed by protein-protein interactions are well-suited for creating enzyme active sites. This discovery has implications for protein evolution and engineering: from an evolutionary perspective, three-coordinated zinc at a homodimer interface cleft represents a simple evolutionary path to nascent enzymatic activity; from a protein engineering perspective, future efforts in de novo design of enzyme active sites may benefit from exploring clefts at protein interfaces for active site placement.

Metal-mediated affinity and orientation specificity in a computationally designed protein homodimer.

Computationally designing protein-protein interactions with high affinity and desired orientation is a challenging task. Incorporating metal-binding sites at the target interface may be one approach for increasing affinity and specifying the binding mode, thereby improving robustness of designed interactions for use as tools in basic research as well as in applications from biotechnology to medicine. Here we describe a Rosetta-based approach for the rational design of a protein monomer to form a zinc-mediated, symmetric homodimer. Our metal interface design, named MID1 (NESG target ID OR37), forms a tight dimer in the presence of zinc (MID1-zinc) with a dissociation constant <30 nM. Without zinc the dissociation constant is 4 μM. The crystal structure of MID1-zinc shows good overall agreement with the computational model, but only three out of four designed histidines coordinate zinc. However, a histidine-to-glutamate point mutation resulted in four-coordination of zinc, and the resulting metal binding site and dimer orientation closely matches the computational model (Cα rmsd = 1.4 Å).

Alternative Computational Protocols for Supercharging Protein Surfaces for Reversible Unfolding and Retention of Stability

Reengineering protein surfaces to exhibit high net charge, referred to as “supercharging”, can improve reversibility of unfolding by preventing aggregation of partially unfolded states. Incorporation of charged side chains should be optimized while considering structural and energetic consequences, as numerous mutations and accumulation of like-charges can also destabilize the native state. A previously demonstrated approach deterministically mutates flexible polar residues (amino acids DERKNQ) with the fewest average neighboring atoms per side chain atom (AvNAPSA). Our approach uses Rosetta-based energy calculations to choose the surface mutations. Both protocols are available for use through the ROSIE web server. The automated Rosetta and AvNAPSA approaches for supercharging choose dissimilar mutations, raising an interesting division in surface charging strategy. Rosetta-supercharged variants of GFP (RscG) ranging from −11 to −61 and +7 to +58 were experimentally tested, and for comparison, we re-tested the previously developed AvNAPSA-supercharged variants of GFP (AscG) with +36 and −30 net charge. Mid-charge variants demonstrated ∼3-fold improvement in refolding with retention of stability. However, as we pushed to higher net charges, expression and soluble yield decreased, indicating that net charge or mutational load may be limiting factors. Interestingly, the two different approaches resulted in GFP variants with similar refolding properties. Our results show that there are multiple sets of residues that can be mutated to successfully supercharge a protein, and combining alternative supercharge protocols with experimental testing can be an effective approach for charge-based improvement to refolding.

Design of Active Transport Must Be Highly Intricate: A Possible Role of Myosin and Ena/VASP for G-Actin Transport in Filopodia

Recent modeling of filopodia--the actin-based cell organelles employed for sensing and motility--reveals that one of the key limiting factors of filopodial length is diffusional transport of G-actin monomers to the polymerizing barbed ends. We have explored the possibility of active transport of G-actin by myosin motors, which would be an expected biological response to overcome the limitation of a diffusion-based process. We found that in a straightforward implementation of active transport the increase in length was unimpressive, < or = 30%, due to sequestering of G-actin by freely diffusing motors. However, artificially removing motor sequestration reactions led to approximately threefold increases in filopodial length, with the transport being mainly limited by the motors failing to detach from the filaments near the tip, clogging the cooperative conveyer belt dynamics. Making motors sterically transparent led to a qualitative change of the dynamics to a different regime of steady growth without a stationary length. Having identified sequestration and clogging as ubiquitous constraints to motor-driven transport, we devised and tested a speculative means to sidestep these limitations in filopodia by employing cross-linking and putative scaffolding roles of Ena/VASP proteins. We conclude that a naïve design of molecular-motor-based active transport would almost always be inefficient--an intricately organized kinetic scheme, with finely tuned rate constants, is required to achieve high-flux transport.

Strategies to control the binding mode of de novo designed protein interactions.

There has been significant recent progress in the computational design of protein interactions including the creation of novel heterodimers, homodimers, nanohedra, fibril caps and a protein crystal. Essential to these successes has been the use of innovative strategies for finding binding modes that are achievable, that is, identifying binding partners and docked conformations that can be successfully stabilized via sequence optimization and backbone refinement. In many cases this has involved the use of structural motifs commonly found at naturally occurring interfaces including alpha helices inserted into hydrophobic grooves, beta-strand pairing, metal binding, established helix packing motifs, and the use of symmetry to form cooperative interactions. Future challenges include the creation of hydrogen bond networks and antibody-like interactions based on the redesign of protein surface loops.

Combined computational design of a zinc-binding site and a protein-protein interaction: one open zinc coordination site was not a robust hotspot for de novo ubiquitin binding.

We computationally designed a de novo protein–protein interaction between wild‐type ubiquitin and a redesigned scaffold. Our strategy was to incorporate zinc at the designed interface to promote affinity and orientation specificity. A large set of monomeric scaffold surfaces were computationally engineered with three‐residue zinc coordination sites, and the ubiquitin residue H68 was docked to the open coordination site to complete a tetrahedral zinc site. This single coordination bond was intended as a hotspot and polar interaction for ubiquitin binding, and surrounding residues on the scaffold were optimized primarily as hydrophobic residues using a rotamer‐based sequence design protocol in Rosetta. From thousands of independent design simulations, four sequences were selected for experimental characterization. The best performing design, called Spelter, binds tightly to zinc (Kd < 10 nM) and binds ubiquitin with a Kd of 20 µM in the presence of zinc and 68 µM in the absence of zinc. Mutagenesis studies and nuclear magnetic resonance chemical shift perturbation experiments indicate that Spelter interacts with H68 and the target surface on ubiquitin; however, H68 does not form a hotspot as intended. Instead, mutation of H68 to alanine results in tighter binding. Although a 3/1 zinc coordination arrangement at an interface cannot be ruled out as a means to improve affinity, our study led us to conclude that 2/2 coordination arrangements or multiple‐zinc designs are more likely to promote high‐affinity protein interactions. Proteins 2013; 81:1245–1255.

Computational de novo design of a four‐helix bundle protein—DND_4HB

The de novo design of proteins is a rigorous test of our understanding of the key determinants of protein structure. The helix bundle is an interesting de novo design model system due to the diverse topologies that can be generated from a few simple α‐helices. Previously, noncomputational studies demonstrated that connecting amphipathic helices together with short loops can sometimes generate helix bundle proteins, regardless of the bundles exact sequence. However, using such methods, the precise positions of helices and side chains cannot be predetermined. Since protein function depends on exact positioning of residues, we examined if sequence design tools in the program Rosetta could be used to design a four‐helix bundle with a predetermined structure. Helix position was specified using a folding procedure that constrained the design model to a defined topology, and iterative rounds of rotamer‐based sequence design and backbone refinement were used to identify a low energy sequence for characterization. The designed protein, DND_4HB, unfolds cooperatively (Tm >90°C) and a NMR solution structure shows that it adopts the target helical bundle topology. Helices 2, 3, and 4 agree very closely with the design model (backbone RMSD = 1.11 Å) and >90% of the core side chain χ1 and χ2 angles are correctly predicted. Helix 1 lies in the target groove against the other helices, but is displaced 3 Å along the bundle axis. This result highlights the potential of computational design to create bundles with atomic‐level precision, but also points at remaining challenges for achieving specific positioning between amphipathic helices.

Cages from coils

The use of coiled coils could facilitate the modular, predictable design of protein nanocages.

From Computational Design to a Protein That Binds

Researchers use computational approaches to design two proteins from scratch that bind to the flu virus. Protein-protein interactions are critical for many biological processes, and over the past several decades, this importance has prompted researchers to investigate the physical and chemical bases of protein binding. How much do we now understand about how proteins interact? Perhaps the most rigorous way to answer this question is to endeavor to design, at an atomic level of detail, such an interaction from scratch. On page 816 of this issue, Fleishman et al. (1) take on this challenge. They used a computational method that enabled them to design two proteins that bind to a preselected surface on an influenza virus. This work demonstrates how far we have come in understanding and being able to predict protein-protein interactions, and the technique could prove useful for developing biosensors, reagents, and more effective drugs.