S. Raguram

Glycomics: a pathway to a class of new and improved therapeutics

Complex glycans that are located at the surface of cells, deposited in the extracellular matrix and attached to soluble signalling molecules have a crucial role in the phenotypic expression of cellular genotypes. However, owing to their structural complexity and some redundancy in terms of structures that elicit a function, the therapeutic potential of complex glycans has not been well exploited, with a few notable exceptions. This review outlines recent advances that promise to increase our ability to use complex glycans as therapeutics. Opportunities for the development of further structure–function relationships for these complex molecules are also discussed.

Quantitative biochemical rationale for differences in transmissibility of 1918 pandemic influenza A viruses

The human adaptation of influenza A viruses is critically governed by the binding specificity of the viral surface hemagglutinin (HA) to long (chain length) α2-6 sialylated glycan (α2-6) receptors on the human upper respiratory tissues. A recent study demonstrated that whereas the 1918 H1N1 pandemic virus, A/South Carolina/1/1918 (SC18), with α2-6 binding preference transmitted efficiently, a single amino acid mutation on HA resulted in a mixed α2-3 sialylated glycan (α2-3)/α2-6 binding virus (NY18) that transmitted inefficiently. To define the biochemical basis for the observed differences in virus transmission, in this study, we have developed an approach to quantify the multivalent HA–glycan interactions. Analysis of the molecular HA–glycan contacts showed subtle changes resulting from the single amino acid variations between SC18 and NY18. The effect of these changes on glycan binding is amplified by multivalency, resulting in quantitative differences in their long α2-6 glycan binding affinities. Furthermore, these differences are also reflected in the markedly distinct binding pattern of SC18 and NY18 HA to the physiological glycans present in human upper respiratory tissues. Thus, the dramatic lower binding affinity of NY18 to long α2-6 glycans, as against a mixed α2-3/6 binding, correlates with its inefficient transmission. In summary, this study establishes a quantitative biochemical correlate for influenza A virus transmission.

Atomic Interaction Networks in the Core of Protein Domains and Their Native Folds

Vastly divergent sequences populate a majority of protein folds. In the quest to identify features that are conserved within protein domains belonging to the same fold, we set out to examine the entire protein universe on a fold-by-fold basis. We report that the atomic interaction network in the solvent-unexposed core of protein domains are fold-conserved, extraordinary sequence divergence notwithstanding. Further, we find that this feature, termed protein core atomic interaction network (or PCAIN) is significantly distinguishable across different folds, thus appearing to be “signature” of a domains native fold. As part of this study, we computed the PCAINs for 8698 representative protein domains from families across the 1018 known protein folds to construct our seed database and an automated framework was developed for PCAIN-based characterization of the protein fold universe. A test set of randomly selected domains that are not in the seed database was classified with over 97% accuracy, independent of sequence divergence. As an application of this novel fold signature, a PCAIN-based scoring scheme was developed for comparative (homology-based) structure prediction, with 1–2 angstroms (mean 1.61A) Cα RMSD generally observed between computed structures and reference crystal structures. Our results are consistent across the full spectrum of test domains including those from recent CASP experiments and most notably in the ‘twilight’ and ‘midnight’ zones wherein <30% and <10% target-template sequence identity prevails (mean twilight RMSD of 1.69A). We further demonstrate the utility of the PCAIN protocol to derive biological insight into protein structure-function relationships, by modeling the structure of the YopM effector novel E3 ligase (NEL) domain from plague-causative bacterium Yersinia Pestis and discussing its implications for host adaptive and innate immune modulation by the pathogen. Considering the several high-throughput, sequence-identity-independent applications demonstrated in this work, we suggest that the PCAIN is a fundamental fold feature that could be a valuable addition to the arsenal of protein modeling and analysis tools.

Redesign of a cross-reactive antibody to dengue virus with broad-spectrum activity and increased in vivo potency

Significance Dengue virus infects more than 200 million people each year, and incidence of severe disease is increasing with no effective countermeasures. We demonstrate in this paper the engineering of an antibody that binds to all four serotypes of dengue virus with potent activity in vitro and in vivo. We also outline a distinct and widely applicable approach to antibody engineering that provides important information on the paratope/epitope interface in the absence of crystal structure data, enabling identification of antibody amino acids that could be mutated. We demonstrate experimentally the alteration of both specificity (enabling cross-serotype binding) and affinity of the engineered antibody. Affinity improvement of proteins, including antibodies, by computational chemistry broadly relies on physics-based energy functions coupled with refinement. However, achieving significant enhancement of binding affinity (>10-fold) remains a challenging exercise, particularly for cross-reactive antibodies. We describe here an empirical approach that captures key physicochemical features common to antigen–antibody interfaces to predict protein–protein interaction and mutations that confer increased affinity. We apply this approach to the design of affinity-enhancing mutations in 4E11, a potent cross-reactive neutralizing antibody to dengue virus (DV), without a crystal structure. Combination of predicted mutations led to a 450-fold improvement in affinity to serotype 4 of DV while preserving, or modestly increasing, affinity to serotypes 1–3 of DV. We show that increased affinity resulted in strong in vitro neutralizing activity to all four serotypes, and that the redesigned antibody has potent antiviral activity in a mouse model of DV challenge. Our findings demonstrate an empirical computational chemistry approach for improving protein–protein docking and engineering antibody affinity, which will help accelerate the development of clinically relevant antibodies.