Thermal Characterization and Interaction of the Subunits from the Multimeric Bacteriophage Endolysin PlyC.

Bacteriophage endolysins degrade the bacterial peptidoglycan and are considered enzymatic alternatives to small-molecule antibiotics. In particular, the multimeric streptococcal endolysin PlyC has appealing antibacterial properties. However, a comprehensive thermal analysis of PlyC is lacking, which is necessary for evaluating its long-term stability and downstream therapeutic potential. Biochemical and kinetic-based methods were used in combination with differential scanning calorimetry to investigate the structural, kinetic, and thermodynamic stability of PlyC and its various subunits and domains. The PlyC holoenzyme structure is irreversibly compromised due to partial unfolding and aggregation at 46 °C. Unfolding of the catalytic subunit, PlyCA, instigates this event, resulting in the kinetic inactivation of the endolysin. In contrast to PlyCA, the PlyCB octamer (the cell wall-binding domain) is thermostable, denaturing at ~75 °C. The isolation of PlyCA or PlyCB alone altered their thermal properties. Contrary to the holoenzyme, PlyCA alone unfolds uncooperatively and is thermodynamically destabilized, whereas the PlyCB octamer reversibly dissociates into monomers and forms an intermediate state at 74 °C in phosphate-buffered saline with each subunit subsequently denaturing at 92 °C. Adding folded PlyCA to an intermediate state PlyCB, followed by cooling, allowed for in vitro reconstitution of the active holoenzyme.

Effects of glycans and hinge on dynamics in the IgG1 Fc.

The crystallizable fragment (Fc) domain of immunoglobulin subclass IgG1 antibodies is engineered for a wide variety of pharmaceutical applications. Two important structural variables in Fc constructs are the hinge region connecting the Fc to the antigen binding fragments (Fab) and the glycans present in various glycoforms. These components affect receptor binding interactions that mediate immune activation. To design new antibody drugs, a robust in silico method for linking stability to structural changes is necessary. In this work, all-atom simulations were used to compare the dynamic behavior of the four structural variants arising from presence or absence of the hinge and glycans. We expressed the simplest of these constructs, the 'minimal Fc' with no hinge and no glycans, in Escherichia coli and report its crystal structure. The 'maximal Fc' that includes full hinge and G0F/G1F glycans is based on a previously reported structure, Protein Data Bank (PDB) ID: 5VGP. These, along with two intermediate structures (with only the glycans or with only the hinge) were used to independently measure the stability effects of the two structural variables using umbrella sampling simulations. Principal component analysis (PCA) was used to determine free energy effects along the Fc's dominant mode of motion. This work provides a comprehensive picture of the effects of hinge and glycans on Fc dynamics and stability.Communicated by Ramaswamy H. Sarma.

Platform development for expression and purification of stable isotope labeled monoclonal antibodies in Escherichia coli.

The widespread use of monoclonal antibodies (mAbs) as a platform for therapeutic drug development in the pharmaceutical industry has led to an increased interest in robust experimental approaches for assessment of mAb structure, stability and dynamics. The ability to enrich proteins with stable isotopes is a prerequisite for the in-depth application of many structural and biophysical methods, including nuclear magnetic resonance (NMR), small angle neutron scattering, neutron reflectometry, and quantitative mass spectrometry. While mAbs can typically be produced with very high yields using mammalian cell expression, stable isotope labeling using cell culture is expensive and often impractical. The most common and cost-efficient approach to label proteins is to express proteins in Escherichia coli grown in minimal media; however, such methods for mAbs have not been reported to date. Here we present, for the first time, the expression and purification of a stable isotope labeled mAb from a genetically engineered E. coli strain capable of forming disulfide bonds in its cytoplasm. It is shown using two-dimensional NMR spectral fingerprinting that the unlabeled mAb and the mAb singly or triply labeled with 13C, 15N, 2H are well folded, with only minor structural differences relative to the mammalian cell-produced mAb that are attributed to the lack of glycosylation in the Fc domain. This advancement of an E. coli-based mAb expression platform will facilitate the production of mAbs for in-depth structural characterization, including the high resolution investigation of mechanisms of action.

2D (1)H(N), (15)N Correlated NMR Methods at Natural Abundance for Obtaining Structural Maps and Statistical Comparability of Monoclonal Antibodies.

PURPOSE: High-resolution nuclear magnetic resonance spectroscopy (NMR) provides a robust approach for producing unique spectral signatures of protein higher order structure at atomic resolution. Such signatures can be used as a tool to establish consistency of protein folding for the assessment of monoclonal antibody (mAb) drug quality and comparability. METHODS: Using the NIST monoclonal antibody (NISTmAb) and a commercial-sourced polyclonal antibody, both IgG1κ isotype, we apply 2D NMR methods at natural abundance for the acquisition and unbiased statistical analysis of (1)H(N) -(15)N correlated spectra of intact antibody (Ab) and protease-cleaved Fab and Fc fragments. RESULTS: The study demonstrates the feasibility of applying 2D NMR techniques to Abs and the precision with which these methods can be used to map structure and establish comparability between samples at atomic resolution. CONCLUSIONS: The statistical analyses suggests that, within the limit of detection, no significant structural differences are observed between the Fab and Fc domains of each respective intact Ab and its corresponding fragments. Discrimination between dissimilar species, such as between the Fab domains of both Abs or between the glycosylated and deglycosylated Fc domains, was further demonstrated. As such, these methods should find general utility for the assessment of mAb higher order structure.

Protein labeling in Escherichia coli with (2)H, (13)C, and (15)N.

A number of structural biology techniques such as nuclear magnetic resonance spectroscopy and small-angle neutron scattering can be performed with proteins with nuclei at natural isotope abundance. However, the use of proteins labeled with stable isotopes ((2)H, (13)C, and (15)N) enables greater experimental flexibility. In this chapter, several methods for uniform and fractional protein labeling with stable isotopes using Escherichia coli in a defined media are described. The methods described can be used for labeling with single or multiple isotopes.

Use of a bacteriophage lysin, PlyC, as an enzyme disinfectant against Streptococcus equi.

Streptococcus equi is the causative agent of the purulent infection equine strangles. This disease is transmitted through shedding of live bacteria from nasal secretions and abscess drainage or by contact with surfaces contaminated by the bacteria. Disinfectants are effective against S. equi, but inactivation by environmental factors, damage to equipment, and toxicity are of great concern. Bacteriophage-encoded lysins (cell wall hydrolases) have been investigated as therapeutic agents due to their ability to lyse susceptible gram-positive organisms. Here, we investigate the use of one lysin, PlyC, as a narrow-spectrum disinfectant against S. equi. This enzyme was active against >20 clinical isolates of S. equi, including both S. equi subsp. equi and S. equi subsp. zooepidemicus. Significantly, PlyC was 1,000 times more active on a per weight basis than Virkon-S, a common disinfecting agent, with 1 microg of enzyme able to sterilize a 10(8) CFU/ml culture of S. equi in 30 min. PlyC was subjected to a standard battery of tests including the Use Dilution Method for Testing Disinfectants and the Germicidal Spray Products Test. Results indicate that aerosolized PlyC can eradicate or significantly reduce the S. equi load on a variety of materials found on common stable and horse-related equipment. Additionally, PlyC was shown to retain full activity under conditions that mimic a horse stable, i.e., in the presence of nonionic detergents, hard water, or organic materials. We propose PlyC as the first protein-based, narrow-spectrum disinfectant against S. equi, which may augment or supplement the use of broad-spectrum disinfectants in barns and stables where equine strangles is prevalent.

Crystal structure of the human carboxypeptidase N (kininase I) catalytic domain.

Human carboxypeptidase N (CPN), a member of the CPN/E subfamily of "regulatory" metallo-carboxypeptidases, is an extracellular glycoprotein synthesized in the liver and secreted into the blood, where it controls the activity of vasoactive peptide hormones, growth factors and cytokines by specifically removing C-terminal basic residues. Normally, CPN circulates in blood plasma as a hetero-tetramer consisting of two 83 kDa (CPN2) domains each flanked by a 48 to 55 kDa catalytic (CPN1) domain. We have prepared and crystallized the recombinant C-terminally truncated catalytic domain of human CPN1, and have determined and refined its 2.1 A crystal structure. The structural analysis reveals that CPN1 has a pear-like shape, consisting of a 319 residue N-terminal catalytic domain and an abutting, cylindrically shaped 79 residue C-terminal beta-sandwich transthyretin (TT) domain, more resembling CPD-2 than CPM. Like these other CPN/E members, two surface loops surrounding the active-site groove restrict access to the catalytic center, offering an explanation for why some larger protein carboxypeptidase inhibitors do not inhibit CPN. Modeling of the Pro-Phe-Arg C-terminal end of the natural substrate bradykinin into the active site shows that the S1' pocket of CPN1 might better accommodate P1'-Lys than Arg residues, in agreement with CPN's preference for cleaving off C-terminal Lys residues. Three Thr residues at the distal TT edge of CPN1 are O-linked to N-acetyl glucosamine sugars; equivalent sites in the membrane-anchored CPM are occupied by basic residues probably involved in membrane interaction. In tetrameric CPN, each CPN1 subunit might interact with the central leucine-rich repeat tandem of the cognate CPN2 subunit via a unique hydrophobic surface patch wrapping around the catalytic domain-TT interface, exposing the two active centers.