Influence of canonical structure determining residues on antibody affinity and stability.

A number of light and heavy chain canonical residue core redesigns were made in a therapeutic antibody (AQC2, anti-VLA1) Fab to explore the consequences to binding affinity and stability. These positions are all loop supporting, primarily CDR1 residues which do not directly contact the antigen. Structure based methods were used with and without consensus sequence information. 30 constructs were made, 24 expressed, and 70% of the designs using consensus sequence information retained binding affinity. Some success maintaining stability with more extreme redesigns suggests a surprising tolerance to mutation, though it often comes at the cost of loss of binding affinity and presumed loop conformation changes. In concordance with the expected need to present an ordered surface for binding, a relationship between decreased affinity and decreased stability was observed. Overpacking the core tends to destabilize the molecule and should be avoided.

A knowledge-based forcefield for protein-protein interface design.

A distance-dependent knowledge-based potential for protein-protein interactions is derived and tested for application in protein design. Information on residue type specific C(alpha) and C(beta) pair distances is extracted from complex crystal structures in the Protein Data Bank and used in the form of radial distribution functions. The use of only backbone and C(beta) position information allows generation of relative protein-protein orientation poses with minimal sidechain information. Further coarse-graining can be done simply in the same theoretical framework to give potentials for residues of known type interacting with unknown type, as in a one-sided interface design problem. Both interface design via pose generation followed by sidechain repacking and localized protein-protein docking tests are performed on 39 nonredundant antibody-antigen complexes for which crystal structures are available. As reference, Lennard-Jones potentials, unspecific for residue type and biasing toward varying degrees of residue pair separation are used as controls. For interface design, the knowledge-based potentials give the best combination of consistently designable poses, low RMSD to the known structure, and more tightly bound interfaces with no added computational cost. 77% of the poses could be designed to give complexes with negative free energies of binding. Generally, larger interface separation promotes designability, but weakens the binding of the resulting designs. A localized docking test shows that the knowledge-based nature of the potentials improves performance and compares respectably with more sophisticated all-atoms potentials.

Affinity enhancement of an in vivo matured therapeutic antibody using structure-based computational design.

Improving the affinity of a high-affinity protein-protein interaction is a challenging problem that has practical applications in the development of therapeutic biomolecules. We used a combination of structure-based computational methods to optimize the binding affinity of an antibody fragment to the I-domain of the integrin VLA1. Despite the already high affinity of the antibody (Kd approximately 7 nM) and the moderate resolution (2.8 A) of the starting crystal structure, the affinity was increased by an order of magnitude primarily through a decrease in the dissociation rate. We determined the crystal structure of a high-affinity quadruple mutant complex at 2.2 A. The structure shows that the design makes the predicted contacts. Structural evidence and mutagenesis experiments that probe a hydrogen bond network illustrate the importance of satisfying hydrogen bonding requirements while seeking higher-affinity mutations. The large and diverse set of interface mutations allowed refinement of the mutant binding affinity prediction protocol and improvement of the single-mutant success rate. Our results indicate that structure-based computational design can be successfully applied to further improve the binding of high-affinity antibodies.

Protein aggregation determinants from a simplified model: cooperative folders resist aggregation.

Two-chain aggregation simulations using minimalist models of proteins G, L, and mutants were used to investigate the fundamentals of protein aggregation. Mutations were selected to break up repeats of hydrophobic beads in the sequence while maintaining native topology and folding ability. Data are collected under conditions in which all chain types have similar folded populations and after equilibrating the separated chains to minimize competition between folding and aggregation. Folding cooperativity stands out as the best single-chain determinant under these conditions and for these simple models. It can be experimentally measured by the width of the unfolding transition during thermal denaturation and loosely related to population of intermediate-like states during folding. Additional measures of cooperativity and other properties such as radius of gyration fluctuations and patterning of hydrophobic residues are also examined. Initial contact system states with transition-state characteristics can be identified and are more expanded than average initial contact states. Two-chain minimalist model aggregates are considerably less structured than their native states and have minimal domain-swapping features.

Influence of denatured and intermediate states of folding on protein aggregation.

We simulate the aggregation thermodynamics and kinetics of proteins L and G, each of which self-assembles to the same alpha/beta [corrected] topology through distinct folding mechanisms. We find that the aggregation kinetics of both proteins at an experimentally relevant concentration exhibit both fast and slow aggregation pathways, although a greater proportion of protein G aggregation events are slow relative to those of found for protein L. These kinetic differences are correlated with the amount and distribution of intrachain contacts formed in the denatured state ensemble (DSE), or an intermediate state ensemble (ISE) if it exists, as well as the folding timescales of the two proteins. Protein G aggregates more slowly than protein L due to its rapidly formed folding intermediate, which exhibits native intrachain contacts spread across the protein, suggesting that certain early folding intermediates may be selected for by evolution due to their protective role against unwanted aggregation. Protein L shows only localized native structure in the DSE with timescales of folding that are commensurate with the aggregation timescale, leaving it vulnerable to domain swapping or nonnative interactions with other chains that increase the aggregation rate. Folding experiments that characterize the structural signatures of the DSE, ISE, or the transition state ensemble (TSE) under nonaggregating conditions should be able to predict regions where interchain contacts will be made in the aggregate, and to predict slower aggregation rates for proteins with contacts that are dispersed across the fold. Since proteins L and G can both form amyloid fibrils, this work also provides mechanistic and structural insight into the formation of prefibrillar species.

An antibody loop replacement design feasibility study and a loop-swapped dimer structure.

A design approach was taken to investigate the feasibility of replacing single complementarity determining region (CDR) antibody loops. This approach may complement simpler mutation-based strategies for rational antibody design by expanding conformation space. Enormous crystal structure diversity is available, making CDR loops logical targets for structure-based design. A detailed analysis for the L1 loop shows that each loop length takes a distinct conformation, thereby allowing control on a length scale beyond that accessible to simple mutations. The L1 loop in the anti-VLA1 antibody was replaced with the L2 loop residues longer in an attempt to add an additional hydrogen bond and fill space on the antibody-antigen interface. The designs expressed well, but failed to improve affinity. In an effort to learn more, one design was crystallized and data were collected at 1.9 A resolution. The designed L1 loop takes the qualitatively desired conformation; confirming that loop replacement by design is feasible. The crystal structure also shows that the outermost loop (residues Leu51-Ser68) is domain swapped with another monomer. Tryptophan fluorescence measurements were used to monitor unfolding as a function of temperature and indicate that the loop involved in domain swapping does not unfold below 60 degrees C. The domain-swapping is not directly responsible for the affinity loss, but is likely a side-effect of the structural instability which may contribute to affinity loss. A second round of design was successful in eliminating the dimerization through mutation of a residue (Leu51Ser) at the joint of the domain-swapped loop.

Trends in antibody sequence changes during the somatic hypermutation process.

Probable germline gene sequences from thousands of aligned mature Ab sequences are inferred using simple computational matching to known V(D)J genes. Comparison of the germline to mature sequences in a structural region-dependent fashion allows insights into the methods that nature uses to mature Abs during the somatic hypermutation process. Four factors determine the residue type mutation patterns: biases in the germline, accessibility from single base permutations, location of mutation hotspots, and functional pressures during selection. Germline repertoires at positions that commonly contact the Ag are biased with tyrosine, serine, and tryptophan. These residue types have a high tendency to be present in mutation hotspot motifs, and their abundance is decreased during maturation by a net conversion to other types. The heavy use of tyrosines on mature Ab interfaces is thus a reflection of the germline composition rather than being due to selection during maturation. Potentially stabilizing changes such as increased proline usage and a small number of double cysteine mutations capable of forming disulfide bonds are ascribed to somatic hypermutation. Histidine is the only residue type for which usage increases in each of the interface, core, and surface regions. The net overall effect is a conversion from residue types that could provide nonspecific initial binding into a diversity of types that improve affinity and stability. Average mutation probabilities are approximately 4% for core residues, approximately 5% for surface residues, and approximately 12% for residues in common Ag-contacting positions, excepting the those coded by the D gene.

Simulated adsorption properties and synthesis prospects of homochiral porous solids based on their heterochiral analogs.

Molecular simulations of chiral molecules in porous heterochiral materials were performed to investigate fundamental adsorption properties and possibilities for production of homochiral porous solids. Zeolite BEA polymorph A and zeotype UCSB-7K each provide separated pores of opposite chirality. Single enantiomer and racemic mixture adsorption results are presented and indicate that significant equilibrium enantiomeric excesses of 40-70% in UCSB-7K and 10% in BEA can be achieved. Larger, better-fitting molecules display higher enantiomeric excesses. For dimethylallene, which moves on molecular dynamics time scales in UCSB-7K, self-diffusivities vary by almost an order of magnitude between the two opposite-handed UCSB-7K pores for a given enantiomer. The predicted properties indicate that equilibrium and nonequilibrium strategies using related homochiral materials for separations may be successful. To this end, a discussion of strategies for selectively blocking pores of one chirality on the basis of enantiomer segregation is provided.

Stable mechanistically-relevant aromatic-based carbenium ions in zeolite catalysts.

The existence of carbenium ion species is assumed in many zeolite catalysis mechanisms. Using computational techniques that include environmental effects, a benzenium-type carbenium ion is identified in zeolite catalysts for the first time. Localization of nearby transition states indicate that this species may play an important role as an intermediate in the bimolecular m-xylene disproportionation reaction. The barrier to back-donation of the proton from the benzenium ion is at least 50 kJ/mol, meaning that this species may be spectroscopically observable. An additional carbenium ion intermediate, formed by abstraction of a hydride from m-xylene, is also predicted. The stability of this second new carbenium ion suggests that aromatic-based carbenium ions are likely to be intermediates in many zeolite-catalyzed reactions. Two types of fundamentally different fully periodic calculations support the stability predictions.

Computational elucidation of the transition state shape selectivity phenomenon.

The most commonly cited example of a transition state shape selective reaction, m-xylene disproportionation in zeolites, is examined to determine if the local spatial environment of a reaction can significantly alter selectivity. In the studied reaction, ZPE-corrected rate limiting energy barriers are 136 kJ/mol for the methoxide-mediated pathway and 109 to 145 kJ/mol for the diphenylmethane-mediated pathway. Both pathways are likely to contribute to selectivity and disfavor one product isomer (1,3,5-trimethylbenzene), but relative selectivity to the other two isomers varies with pore geometry, mechanistic pathway, and inclusion of entropic effects. Most importantly, study of one pathway in three different common zeolite framework types (FAU, MFI, and MOR) allows explicit and practically oriented consideration of pore shape. Variation of the environment shape at the critical transition states is thus shown to affect the course of reaction. Barrier height shifts on the order of 10-20 kJ/mol are achievable. Observed selectivities do not agree with the transition state characteristics calculated here and, hence, are most likely due to product shape selectivity. Further examination of the pathways highlights the importance of mechanistic steps that do not result in isomer-defining bonds and leads to a more robust definition of transition state shape selectivity.