BindCraft: one-shot design of functional protein binders.

Protein-protein interactions (PPIs) are at the core of all key biological processes. However, the complexity of the structural features that determine PPIs makes their design challenging. We present BindCraft, an open-source and automated pipeline for protein binder design with experimental success rates of 10-100%.

A Bis(imidazole)-based cysteine labeling tool for metalloprotein assembly.

Precise metal-protein coordination by design remains a considerable challenge. Polydentate, high-metal-affinity protein modifications, both chemical and recombinant, can enable metal localization. However, these constructs are often bulky, conformationally and stereochemically ill-defined, or coordinately saturated.

Massively parallel, computationally guided design of a proenzyme.

Confining the activity of a designed protein to a specific microenvironment would have broad-ranging applications, such as enabling cell type-specific therapeutic action by enzymes while avoiding off-target effects. While many natural enzymes are synthesized as inactive zymogens that can be activated by proteolysis, it has been challenging to redesign any chosen enzyme to be similarly stimulus responsive. Here, we develop a massively parallel computational design, screening, and next-generation sequencing-based approach for proenzyme design.

Ensuring scientific reproducibility in bio-macromolecular modeling via extensive, automated benchmarks.

Each year vast international resources are wasted on irreproducible research. The scientific community has been slow to adopt standard software engineering practices, despite the increases in high-dimensional data, complexities of workflows, and computational environments. Here we show how scientific software applications can be created in a reproducible manner when simple design goals for reproducibility are met.

MHCEpitopeEnergy, a Flexible Rosetta-Based Biotherapeutic Deimmunization Platform.

As non-"self" macromolecules, biotherapeutics can trigger an immune response that can reduce drug efficacy, require patients to be taken off therapy, or even cause life-threatening reactions. To enable the flexible and facile design of protein biotherapeutics while reducing the prevalence of T-cell epitopes that drive immune recognition, we have integrated into the Rosetta protein design suite a new scoring term that allows design protocols to account for predicted or experimentally identified epitopes in the optimized objective function. This flexible scoring term can be used in any Rosetta design trajectory, can be targeted to specific regions of a protein, and can be readily extended to work with a variety of epitope predictors.

Identification and characterization of hippuristanol-resistant mutants reveals eIF4A1 dependencies within mRNA 5' leader regions.

Hippuristanol (Hipp) is a natural product that selectively inhibits protein synthesis by targeting eukaryotic initiation factor (eIF) 4A, a DEAD-box RNA helicase required for ribosome recruitment to mRNA templates. Hipp binds to the carboxyl-terminal domain of eIF4A, locks it in a closed conformation, and inhibits its RNA binding. The dependencies of mRNAs for eIF4A during initiation is contingent on the degree of secondary structure within their 5' leader region.

Structure-Based Design of Dimeric Bisbenzimidazole Inhibitors to an Emergent Trimethoprim-Resistant Type II Dihydrofolate Reductase Guides the Design of Monomeric Analogues.

The worldwide use of the broad-spectrum antimicrobial trimethoprim (TMP) has induced the rise of TMP-resistant microorganisms. In addition to resistance-causing mutations of the microbial chromosomal dihydrofolate reductase (Dfr), the evolutionarily and structurally unrelated type II Dfrs (DfrBs) have been identified in TMP-resistant microorganisms. DfrBs are intrinsically TMP-resistant and allow bacterial proliferation when the microbial chromosomal Dfr is TMP-inhibited, making these enzymes important targets for inhibitor development.

Computational Design of a Photocontrolled Cytosine Deaminase.

There is growing interest in designing spatiotemporal control over enzyme activities using noninvasive stimuli such as light. Here, we describe a structure-based, computation-guided predictive method for reversibly controlling enzyme activity using covalently attached photoresponsive azobenzene groups. Applying the method to the therapeutically useful enzyme yeast cytosine deaminase, we obtained a ∼3-fold change in enzyme activity by the photocontrolled modulation of the enzyme's active site lid structure, while fully maintaining thermostability.

Engineering carboxypeptidase G2 circular permutations for the design of an autoinhibited enzyme.

Carboxypeptidase G2 (CPG2) is an Food and Drug Administration (FDA)-approved enzyme drug used to treat methotrexate (MTX) toxicity in cancer patients receiving MTX treatment. It has also been used in directed enzyme-prodrug chemotherapy, but this strategy has been hampered by off-site activation of the prodrug by the circulating enzyme. The development of a tumor protease activatable CPG2, which could be achieved using a circular permutation of CPG2 fused to an inactivating 'prodomain', would aid in these applications.

The role of conformational flexibility in Baeyer-Villiger monooxygenase catalysis and structure.

Background: The Baeyer-Villiger monooxygenases (BMVOs) are a group of microbial enzymes that have garnered interest as industrial biocatalysts. While great strides have been made in recent years to understand the mechanism of these enzymes from a structural perspective, our understanding remains incomplete. In particular, the role of a twenty residue loop (residues 487-504), which we refer to as the "Control Loop," that is observed in either an ordered or disordered state in various crystal structures remains unclear.

Comprehensive characterization of ligand-induced plasticity changes in a dimeric enzyme.

Unlabelled: An enzyme's inherent structural plasticity is frequently associated with substrate binding, yet detailed structural characterization of flexible proteins remains challenging. This study employs complementary biophysical methods to characterize the partially unfolded structure of substrate-free AAC(6')-Ii, an N-acetyltransferase of the GCN5-related N-acetyltransferase (GNAT) superfamily implicated in conferring broad-spectrum aminoglycoside resistance on Enterococcus faecium. The X-ray crystal structure of AAC(6')-Ii is analyzed to identify relative motions of the structural elements that constitute the dimeric enzyme.

Lactone-bound structures of cyclohexanone monooxygenase provide insight into the stereochemistry of catalysis.

The Baeyer-Villiger monooxygenases (BVMOs) are microbial enzymes that catalyze the synthetically useful Baeyer-Villiger oxidation reaction. The available BVMO crystal structures all lack a substrate or product bound in a position that would determine the substrate specificity and stereospecificity of the enzyme. Here, we report two crystal structures of cyclohexanone monooxygenase (CHMO) with its product, ε-caprolactone, bound: the CHMO(Tight) and CHMO(Loose) structures.

The substrate-bound crystal structure of a Baeyer-Villiger monooxygenase exhibits a Criegee-like conformation.

The Baeyer-Villiger monooxygenases (BVMOs) are a family of bacterial flavoproteins that catalyze the synthetically useful Baeyer-Villiger oxidation reaction. This involves the conversion of ketones into esters or cyclic ketones into lactones by introducing an oxygen atom adjacent to the carbonyl group. The BVMOs offer exquisite regio- and enantiospecificity while acting on a wide range of substrates.

Novel crystallization conditions for tandem variant R67 DHFR yield a wild-type crystal structure.

Trimethoprim is an antibiotic that targets bacterial dihydrofolate reductase (DHFR). A plasmid-encoded DHFR known as R67 DHFR provides resistance to trimethoprim in bacteria. To better understand the mechanism of this homotetrameric enzyme, a tandem dimer construct was created that linked two monomeric R67 DHFR subunits together and mutated the sequence of residues 66-69 of the first subunit from VQIY to INSF.

Multiple conformers in active site of human dihydrofolate reductase F31R/Q35E double mutant suggest structural basis for methotrexate resistance.

Methotrexate is a slow, tight-binding, competitive inhibitor of human dihydrofolate reductase (hDHFR), an enzyme that provides key metabolites for nucleotide biosynthesis. In an effort to better characterize ligand binding in drug resistance, we have previously engineered hDHFR variant F31R/Q35E. This variant displays a >650-fold decrease in methotrexate affinity, while maintaining catalytic activity comparable to the native enzyme.

Crystal structures of cyclohexanone monooxygenase reveal complex domain movements and a sliding cofactor.

Cyclohexanone monooxygenase (CHMO) is a flavoprotein that carries out the archetypical Baeyer-Villiger oxidation of a variety of cyclic ketones into lactones. Using NADPH and O(2) as cosubstrates, the enzyme inserts one atom of oxygen into the substrate in a complex catalytic mechanism that involves the formation of a flavin-peroxide and Criegee intermediate. We present here the atomic structures of CHMO from an environmental Rhodococcus strain bound with FAD and NADP(+) in two distinct states, to resolutions of 2.

Characterization of the redox and metal binding activity of BsSco, a protein implicated in the assembly of cytochrome c oxidase.

Members of the Sco protein family are implicated in the assembly of the respiratory complex cytochrome c oxidase. Several possible roles have been proposed for Sco: a copper delivery agent, a site-specific thiol reductase, and an indicator of cellular redox status. Two cysteine residues (C45 and C49) in the sequence CXXXCP and a histidine (H135) approximately 90 residues toward the C-terminus are conserved in Sco from bacteria, yeast, and humans.