Protein engineering and the use of molecular modeling and simulation: the case of heterodimeric Fc engineering.

Computational and structure guided methods can make significant contributions to the development of solutions for difficult protein engineering problems, including the optimization of next generation of engineered antibodies. In this paper, we describe a contemporary industrial antibody engineering program, based on hypothesis-driven in silico protein optimization method. The foundational concepts and methods of computational protein engineering are discussed, and an example of a computational modeling and structure-guided protein engineering workflow is provided for the design of best-in-class heterodimeric Fc with high purity and favorable biophysical properties.

Improving biophysical properties of a bispecific antibody scaffold to aid developability: quality by molecular design.

While the concept of Quality-by-Design is addressed at the upstream and downstream process development stages, we questioned whether there are advantages to addressing the issues of biologics quality early in the design of the molecule based on fundamental biophysical characterization, and thereby reduce complexities in the product development stages. Although limited number of bispecific therapeutics are in clinic, these developments have been plagued with difficulty in producing materials of sufficient quality and quantity for both preclinical and clinical studies. The engineered heterodimeric Fc is an industry-wide favorite scaffold for the design of bispecific protein therapeutics because of its structural, and potentially pharmacokinetic, similarity to the natural antibody.

LC-MS characterization and purity assessment of a prototype bispecific antibody.

Bispecific IgG asymmetric (heterodimeric) antibodies offer enhanced therapeutic efficacy, but present unique challenges for drug development. These challenges are related to the proper assembly of heavy and light chains. Impurities such as symmetric (homodimeric) antibodies can arise with improper assembly.

Studies of base pair sequence effects on DNA solvation based on all-atom molecular dynamics simulations.

Detailed analyses of the sequence-dependent solvation and ion atmosphere of DNA are presented based on molecular dynamics (MD) simulations on all the 136 unique tetranucleotide steps obtained by the ABC consortium using the AMBER suite of programs. Significant sequence effects on solvation and ion localization were observed in these simulations. The results were compared to essentially all known experimental data on the subject.

Human subtilase SKI-1/S1P is a master regulator of the HCV Lifecycle and a potential host cell target for developing indirect-acting antiviral agents.

HCV infection is a major risk factor for liver cancer and liver transplantation worldwide. Overstimulation of host lipid metabolism in the liver by HCV-encoded proteins during viral infection creates a favorable environment for virus propagation and pathogenesis. In this study, we hypothesize that targeting cellular enzymes acting as master regulators of lipid homeostasis could represent a powerful approach to developing a novel class of broad-spectrum antivirals against infection associated with human Flaviviridae viruses such as hepatitis C virus (HCV), whose assembly and pathogenesis depend on interaction with lipid droplets (LDs).

Prokaryotic gene finding based on physicochemical characteristics of codons calculated from molecular dynamics simulations.

An ab initio model for gene prediction in prokaryotic genomes is proposed based on physicochemical characteristics of codons calculated from molecular dynamics (MD) simulations. The model requires a specification of three calculated quantities for each codon: the double-helical trinucleotide base pairing energy, the base pair stacking energy, and an index of the propensity of a codon for protein-nucleic acid interactions. The base pairing and stacking energies for each codon are obtained from recently reported MD simulations on all unique tetranucleotide steps, and the third parameter is assigned based on the conjugate rule previously proposed to account for the wobble hypothesis with respect to degeneracies in the genetic code.

Spectroscopic and molecular dynamics evidence for a sequential mechanism for the A-to-B transition in DNA.

The A-to-B form transition has been examined in three DNA duplexes, d(CGCGAATTCGCG)(2), d(CGCGAATTGCGC), and d(CGCAAATTTCGC), using circular dichroism spectroscopy, ultraviolet resonance Raman (UVRR) spectroscopy, and molecular dynamics (MD) simulation. Circular dichroism spectra confirm that these molecules adopt the A form under conditions of reduced water activity. UVRR results, obtained under similar conditions, suggest that the transition involves a series of intermediate forms between A and B.

CTL recognition of a protective immunodominant influenza A virus nucleoprotein epitope utilizes a highly restricted Vbeta but diverse Valpha repertoire: functional and structural implications.

To investigate protective immunity conferred by CTL against viral pathogens, we have analyzed CD8(+) T cell responses to the immunodominant nucleoprotein epitope (NP(366-374)) of influenza A virus in B6 mice during primary and secondary infections in vivo. Unlike the highly biased TCR Vbeta repertoire, the associated Valpha repertoire specific for the NP(366-374)/D(b) ligand is quite diverse. Nonetheless, certain public and conserved CDR3alpha clonotypes with distinct molecular signatures were identified.

Root mean square deviation probability analysis of molecular dynamics trajectories on DNA.

The comparison and detection of the commonalities and differences in multiple structural ensembles is an important step in the use of molecular simulations to gain insight into the conformation and dynamics of complex biomacromolecules. While the average structure is often employed as the representative of an ensemble of structures in such comparisons, dynamic molecular systems with multiple conformational substates call for a more accurate representation that captures the complete dynamical range of the ensemble. We present a probability analysis procedure based on the root-mean-square differences among the structural ensembles that efficiently and accurately performs the relevant comparison.

Structural bioinformatics of DNA: a web-based tool for the analysis of molecular dynamics results and structure prediction.

Unlabelled: We report here the release of a web-based tool (MDDNA) to study and model the fine structural details of DNA on the basis of data extracted from a set of molecular dynamics (MD) trajectories of DNA sequences involving all the unique tetranucleotides. The dynamic web interface can be employed to analyze the first neighbor sequence context effects on the 10 unique dinucleotide steps of DNA. Functionality is included to build all atom models of any user-defined sequence based on the MD results.

Can free energy calculations be fast and accurate at the same time? Binding of low-affinity, non-peptide inhibitors to the SH2 domain of the src protein.

The usefulness of free-energy calculations in non-academic environments, in general, and in the pharmaceutical industry, in particular, is a long-time debated issue, often considered from the angle of cost/performance criteria. In the context of the rational drug design of low-affinity, non-peptide inhibitors to the SH2 domain of the (pp60)src tyrosine kinase, the continuing difficulties encountered in an attempt to obtain accurate free-energy estimates are addressed. free-energy calculations can provide a convincing answer, assuming that two key-requirements are fulfilled: (i) thorough sampling of the configurational space is necessary to minimize the statistical error, hence raising the question: to which extent can we sacrifice the computational effort, yet without jeopardizing the precision of the free-energy calculation? (ii) the sensitivity of binding free-energies to the parameters utilized imposes an appropriate parametrization of the potential energy function, especially for non-peptide molecules that are usually poorly described by multipurpose macromolecular force fields.

Molecular dynamics simulations of the 136 unique tetranucleotide sequences of DNA oligonucleotides. II: sequence context effects on the dynamical structures of the 10 unique dinucleotide steps.

Molecular dynamics (MD) simulations including water and counterions on B-DNA oligomers containing all 136 unique tetranucleotide basepair steps are reported. The objective is to obtain the calculated dynamical structure for at least two copies of each case, use the results to examine issues with regard to convergence and dynamical stability of MD on DNA, and determine the significance of sequence context effects on all unique dinucleotide steps. This information is essential to understand sequence effects on DNA structure and has implications on diverse problems in the structural biology of DNA.

Induced fit and the entropy of structural adaptation in the complexation of CAP and lambda-repressor with cognate DNA sequences.

Molecular dynamics (MD) simulations of 5 ns on protein-DNA complexes of catabolite-activator protein (CAP), lambda-repressor, and their corresponding uncomplexed protein and DNA, are reported. These cases represent two extremes of DNA bending, with CAP DNA bent severely and the lambda-operator nearly straight when complexed with protein. The calculations were performed using the AMBER suite of programs and the parm94 force field, validated for these studies by good agreement with experimental nuclear magnetic resonance data on DNA.

Axis curvature and ligand induced bending in the CAP-DNA oligomers.

The origin of DNA axis curvature in complexes of the catabolite activator protein with DNA is studied using multiple molecular dynamics (MD) simulations of the free and protein-bound forms of the DNA. The results are compared to available solution and crystal structure data. The MD simulations reproduce the experimentally observed bend in DNA and indicate that ∼40% of the bending observed in the complex is intrinsic to the DNA sequence, whereas ∼60% is induced on protein binding.

Structure and axis curvature in two dA6 x dT6 DNA oligonucleotides: comparison of molecular dynamics simulations with results from crystallography and NMR spectroscopy.

Molecular dynamics (MD) simulations have been performed on the A6 containing DNA dodecamers d(GGCAAAAAACGG) solved by NMR and d(CGCAAAAAAGCG) solved by crystallography. The experimental structures differ in the direction of axis bending and in other small but important aspects relevant to the DNA curvature problem. Five nanosecond MD simulations of each sequence have been performed, beginning with both the NMR and crystal forms as well as canonical B-form DNA.

Molecular dynamics simulations of the 136 unique tetranucleotide sequences of DNA oligonucleotides. I. Research design and results on d(CpG) steps.

We describe herein a computationally intensive project aimed at carrying out molecular dynamics (MD) simulations including water and counterions on B-DNA oligomers containing all 136 unique tetranucleotide base sequences. This initiative was undertaken by an international collaborative effort involving nine research groups, the "Ascona B-DNA Consortium" (ABC). Calculations were carried out on the 136 cases imbedded in 39 DNA oligomers with repeating tetranucleotide sequences, capped on both ends by GC pairs and each having a total length of 15 nucleotide pairs.

Molecular dynamics simulations of DNA curvature and flexibility: helix phasing and premelting.

Recent studies of DNA axis curvature and flexibility based on molecular dynamics (MD) simulations on DNA are reviewed. The MD simulations are on DNA sequences up to 25 base pairs in length, including explicit consideration of counterions and waters in the computational model. MD studies are described for ApA steps, A-tracts, for sequences of A-tracts with helix phasing.