Jacob E. Corn

Rosetta3: An Object-Oriented Software Suite for the Simulation and Design of Macromolecules

We have recently completed a full re-architecturing of the ROSETTA molecular modeling program, generalizing and expanding its existing functionality. The new architecture enables the rapid prototyping of novel protocols by providing easy-to-use interfaces to powerful tools for molecular modeling. The source code of this rearchitecturing has been released as ROSETTA3 and is freely available for academic use. At the time of its release, it contained 470,000 lines of code. Counting currently unpublished protocols at the time of this writing, the source includes 1,285,000 lines. Its rapid growth is a testament to its ease of use. This chapter describes the requirements for our new architecture, justifies the design decisions, sketches out central classes, and highlights a few of the common tasks that the new software can perform.

Computational design of proteins targeting the conserved stem region of influenza hemagglutinin.

Proteins can be designed that bind to specific patches on target proteins to alter their subsequent interactions. We describe a general computational method for designing proteins that bind a surface patch of interest on a target macromolecule. Favorable interactions between disembodied amino acid residues and the target surface are identified and used to anchor de novo designed interfaces. The method was used to design proteins that bind a conserved surface patch on the stem of the influenza hemagglutinin (HA) from the 1918 H1N1 pandemic virus. After affinity maturation, two of the designed proteins, HB36 and HB80, bind H1 and H5 HAs with low nanomolar affinity. Further, HB80 inhibits the HA fusogenic conformational changes induced at low pH. The crystal structure of HB36 in complex with 1918/H1 HA revealed that the actual binding interface is nearly identical to that in the computational design model. Such designed binding proteins may be useful for both diagnostics and therapeutics.

A prudent path forward for genomic engineering and germline gene modification

A framework for open discourse on the use of CRISPR-Cas9 technology to manipulate the human genome is urgently needed Genome engineering technology offers unparalleled potential for modifying human and nonhuman genomes. In humans, it holds the promise of curing genetic disease, while in other organisms it provides methods to reshape the biosphere for the benefit of the environment and human societies. However, with such enormous opportunities come unknown risks to human health and well-being. In January, a group of interested stakeholders met in Napa, California (1), to discuss the scientific, medical, legal, and ethical implications of these new prospects for genome biology. The goal was to initiate an informed discussion of the uses of genome engineering technology, and to identify those areas where action is essential to prepare for future developments. The meeting identified immediate steps to take toward ensuring that the application of genome engineering technology is performed safely and ethically.

Enhancing homology-directed genome editing by catalytically active and inactive CRISPR-Cas9 using asymmetric donor DNA

Targeted genomic manipulation by Cas9 can efficiently generate knockout cells and organisms via error-prone nonhomologous end joining (NHEJ), but the efficiency of precise sequence replacement by homology-directed repair (HDR) is substantially lower. Here we investigate the interaction of Cas9 with target DNA and use our findings to improve HDR efficiency. We show that dissociation of Cas9 from double-stranded DNA (dsDNA) substrates is slow (lifetime ∼6 h) but that, before complete dissociation, Cas9 asymmetrically releases the 3′ end of the cleaved DNA strand that is not complementary to the sgRNA (nontarget strand). By rationally designing single-stranded DNA (ssDNA) donors of the optimal length complementary to the strand that is released first, we increase the rate of HDR in human cells when using Cas9 or nickase variants to up to 60%. We also demonstrate HDR rates of up to 0.7% using a catalytically inactive Cas9 mutant (dCas9), which binds DNA without cleaving it.

Rosettascripts: A scripting language interface to the Rosetta Macromolecular modeling suite

Macromolecular modeling and design are increasingly useful in basic research, biotechnology, and teaching. However, the absence of a user-friendly modeling framework that provides access to a wide range of modeling capabilities is hampering the wider adoption of computational methods by non-experts. RosettaScripts is an XML-like language for specifying modeling tasks in the Rosetta framework. RosettaScripts provides access to protocol-level functionalities, such as rigid-body docking and sequence redesign, and allows fast testing and deployment of complex protocols without need for modifying or recompiling the underlying C++ code. We illustrate these capabilities with RosettaScripts protocols for the stabilization of proteins, the generation of computationally constrained libraries for experimental selection of higher-affinity binding proteins, loop remodeling, small-molecule ligand docking, design of ligand-binding proteins, and specificity redesign in DNA-binding proteins.

A De Novo Protein Binding Pair By Computational Design and Directed Evolution

The de novo design of protein-protein interfaces is a stringent test of our understanding of the principles underlying protein-protein interactions and would enable unique approaches to biological and medical challenges. Here we describe a motif-based method to computationally design protein-protein complexes with native-like interface composition and interaction density. Using this method we designed a pair of proteins, Prb and Pdar, that heterodimerize with a Kd of 130 nM, 1000-fold tighter than any previously designed de novo protein-protein complex. Directed evolution identified two point mutations that improve affinity to 180 pM. Crystal structures of an affinity-matured complex reveal binding is entirely through the designed interface residues. Surprisingly, in the in vitro evolved complex one of the partners is rotated 180° relative to the original design model, yet still maintains the central computationally designed hotspot interaction and preserves the character of many peripheral interactions. This work demonstrates that high-affinity protein interfaces can be created by designing complementary interaction surfaces on two noninteracting partners and underscores remaining challenges.

Replication origin recognition and deformation by a heterodimeric archaeal orc1 complex

The faithful duplication of genetic material depends on essential DNA replication initiation factors. Cellular initiators form higher-order assemblies on replication origins, using adenosine triphosphate (ATP) to locally remodel duplex DNA and facilitate proper loading of synthetic replisomal components. To better understand initiator function, we determined the 3.4 angstrom–resolution structure of an archaeal Cdc6/Orc1 heterodimer bound to origin DNA. The structure demonstrates that, in addition to conventional DNA binding elements, initiators use their AAA+ ATPase domains to recognize origin DNA. Together these interactions establish the polarity of initiator assembly on the origin and induce substantial distortions into origin DNA strands. Biochemical and comparative analyses indicate that AAA+/DNA contacts observed in the structure are dynamic and evolutionarily conserved, suggesting that the complex forms a core component of the basal initiation machinery.

Community-wide assessment of protein-interface modeling suggests improvements to design methodology

The CAPRI (Critical Assessment of Predicted Interactions) and CASP (Critical Assessment of protein Structure Prediction) experiments have demonstrated the power of community-wide tests of methodology in assessing the current state of the art and spurring progress in the very challenging areas of protein docking and structure prediction. We sought to bring the power of community-wide experiments to bear on a very challenging protein design problem that provides a complementary but equally fundamental test of current understanding of protein-binding thermodynamics. We have generated a number of designed protein-protein interfaces with very favorable computed binding energies but which do not appear to be formed in experiments, suggesting that there may be important physical chemistry missing in the energy calculations. A total of 28 research groups took up the challenge of determining what is missing: we provided structures of 87 designed complexes and 120 naturally occurring complexes and asked participants to identify energetic contributions and/or structural features that distinguish between the two sets. The community found that electrostatics and solvation terms partially distinguish the designs from the natural complexes, largely due to the nonpolar character of the designed interactions. Beyond this polarity difference, the community found that the designed binding surfaces were, on average, structurally less embedded in the designed monomers, suggesting that backbone conformational rigidity at the designed surface is important for realization of the designed function. These results can be used to improve computational design strategies, but there is still much to be learned; for example, one designed complex, which does form in experiments, was classified by all metrics as a nonbinder.

Selection-free genome editing of the sickle mutation in human adult hematopoietic stem/progenitor cells

Hematopoietic stem cells from patients with sickle cell disease can be edited by CRISPR/Cas9 and maintain the edits in vivo. Hammering out the sickle cell mutation Sickle cell disease is a genetic disorder caused by a mutation in one of the hemoglobin genes, which causes deformation of red blood cells and results in occlusion of blood vessels, severe pain crises, and progressive organ injury. To correct the mutation that causes this disease, DeWitt et al. modified hematopoietic stem cells from sickle cell disease patients using a CRISPR/Cas9 gene editing approach. The authors showed that the corrected cells successfully engrafted in a mouse model and produced enough normal hemoglobin to have a potential clinical benefit in the setting of sickle cell disease. Genetic diseases of blood cells are prime candidates for treatment through ex vivo gene editing of CD34+ hematopoietic stem/progenitor cells (HSPCs), and a variety of technologies have been proposed to treat these disorders. Sickle cell disease (SCD) is a recessive genetic disorder caused by a single-nucleotide polymorphism in the β-globin gene (HBB). Sickle hemoglobin damages erythrocytes, causing vasoocclusion, severe pain, progressive organ damage, and premature death. We optimize design and delivery parameters of a ribonucleoprotein (RNP) complex comprising Cas9 protein and unmodified single guide RNA, together with a single-stranded DNA oligonucleotide donor (ssODN), to enable efficient replacement of the SCD mutation in human HSPCs. Corrected HSPCs from SCD patients produced less sickle hemoglobin RNA and protein and correspondingly increased wild-type hemoglobin when differentiated into erythroblasts. When engrafted into immunocompromised mice, ex vivo treated human HSPCs maintain SCD gene edits throughout 16 weeks at a level likely to have clinical benefit. These results demonstrate that an accessible approach combining Cas9 RNP with an ssODN can mediate efficient HSPC genome editing, enables investigator-led exploration of gene editing reagents in primary hematopoietic stem cells, and suggests a path toward the development of new gene editing treatments for SCD and other hematopoietic diseases.

Automated electron-density sampling reveals widespread conformational polymorphism in proteins

Although proteins populate large structural ensembles, X‐ray diffraction data are traditionally interpreted using a single model. To search for evidence of alternate conformers, we developed a program, Ringer, which systematically samples electron density around the dihedral angles of protein side chains. In a diverse set of 402 structures, Ringer identified weak, nonrandom electron‐density features that suggest of the presence of hidden, lowly populated conformations for >18% of uniquely modeled residues. Although these peaks occur at electron‐density levels traditionally regarded as noise, statistically significant (P < 10−5) enrichment of peaks at successive rotameric χ angles validates the assignment of these features as unmodeled conformations. Weak electron density corresponding to alternate rotamers also was detected in an accurate electron density map free of model bias. Ringer analysis of the high‐resolution structures of free and peptide‐bound calmodulin identified shifts in ensembles and connected the alternate conformations to ligand recognition. These results show that the signal in high‐resolution electron density maps extends below the traditional 1 σ cutoff, and crystalline proteins are more polymorphic than current crystallographic models. Ringer provides an objective, systematic method to identify previously undiscovered alternate conformations that can mediate protein folding and function.