With the advent of biosimilar version of brand biologics, regulatory authorities in all major jurisdictions throughout the world have developed guidance documents to facilitate their approval [1][2][3][4] . The common theme in the new guidance documents is the stipulation that sponsors must demonstrate biosimilarity between their proposed product and an approved reference product by using state-of-the-art analytical technologies to reduce the size of clinical studies. In the US guidance documents a "totality of evidence" approach is described that can be used to establish the degree of similarity and guide regulatory decision making 3, 4 .Higher order structure is an important quality attribute of biosimilars that must be assessed in a thorough comparability exercise. To date, the higher order structure has been evaluated with low-resolution techniques, such as circular dichroism, Fourier transform infrared and Raman spectroscopies, and indirectly with several biological and stability assays 5 . In 2008, a two dimensional nuclear magnetic resonance (2D-NMR) spectroscopy approach was first applied to the high-resolution assessment of the higher order structure of a native Correspondence should be addressed to
The increased interest in using monoclonal antibodies (mAbs) as a platform for biopharmaceuticals has led to the need for new analytical techniques that can precisely assess physicochemical properties of these large and very complex drugs for the purpose of correctly identifying quality attributes (QA). One QA, higher order structure (HOS), is unique to biopharmaceuticals and essential for establishing consistency in biopharmaceutical manufacturing, detecting process-related variations from manufacturing changes and establishing comparability between biologic products. To address this measurement challenge, two-dimensional nuclear magnetic resonance spectroscopy (2D-NMR) methods were introduced that allow for the precise atomic-level comparison of the HOS between two proteins, including mAbs. Here, an inter-laboratory comparison involving 26 industrial, government and academic laboratories worldwide was performed as a benchmark using the NISTmAb, from the National Institute of Standards and Technology (NIST), to facilitate the translation of the 2D-NMR method into routine use for biopharmaceutical product development. Two-dimensional 1H,15N and 1H,13C NMR spectra were acquired with harmonized experimental protocols on the unlabeled Fab domain and a uniformly enriched-15N, 20%-13C-enriched system suitability sample derived from the NISTmAb. Chemometric analyses from over 400 spectral maps acquired on 39 different NMR spectrometers ranging from 500 MHz to 900 MHz demonstrate spectral fingerprints that are fit-for-purpose for the assessment of HOS. The 2D-NMR method is shown to provide the measurement reliability needed to move the technique from an emerging technology to a harmonized, routine measurement that can be generally applied with great confidence to high precision assessments of the HOS of mAb-based biotherapeutics.
MinE is required for the dynamic oscillation of Min proteins that restricts formation of the cytokinetic septum to the midpoint of the cell in gram negative bacteria. Critical for this oscillation is MinD-binding by MinE to stimulate MinD ATP hydrolysis, a function that had been assigned to the first ∼30 residues in MinE. Previous models based on the structure of an autonomously folded dimeric C-terminal fragment suggested that the N-terminal domain is freely accessible for interactions with MinD. We report here the solution NMR structure of the full-length MinE dimer from Neisseria gonorrhoeae, with two parts of the N-terminal domain forming an integral part of the dimerization interface. Unexpectedly, solvent accessibility is highly restricted for residues that were previously hypothesized to directly interact with MinD. To delineate the true MinD-binding region, in vitro assays for MinE-stimulated MinD activity were performed. The relative MinD-binding affinities obtained for full-length and N-terminal peptides from MinE demonstrated that residues that are buried in the dimeric interface nonetheless participate in direct interactions with MinD. According to results from NMR spin relaxation experiments, access to these buried residues may be facilitated by the presence of conformational exchange. We suggest that this concealment of MinD-binding residues by the MinE dimeric interface provides a mechanism for prevention of nonspecific interactions, particularly with the lipid membrane, to allow the free diffusion of MinE that is critical for Min protein oscillation.protein structure | conformational dynamics | protein-protein interactions | ATPase
Rhomboid proteins comprise the largest class of intramembrane protease known, being conserved from bacteria to humans. The functional status of these proteases is typically assessed through direct or indirect detection of peptide cleavage products. Although these assays can report on the ability of a rhomboid to catalyze peptide bond cleavage, differences in measured hydrolysis rates can reflect changes in the structure and activity of catalytic residues, as well as the ability of the substrate to access the active site. Here we show that a highly reactive and sterically unencumbered fluorophosphonate activity-based protein profiling probe can be used to report on the catalytic integrity of active site residues in the Escherichia coli GlpG protein. We used results obtained with this probe on GlpG in proteomic samples, in combination with a conventional assay of proteolytic function on purified samples, to identify residues that are located on the cytoplasmic side of the lipid bilayer that are required for maximal proteolytic activity. Regions tested include the 90-residue aqueous-exposed N-terminus that encompasses a globular structure that we have determined by solution nuclear magnetic resonance, along with residues on the cytoplasmic side of the transmembrane domain core. While in most cases mutation or elimination of these residues did not significantly alter the catalytic status of the GlpG active site, the lipid-facing residue Arg227 was found to be important for maintaining a catalytically competent active site. In addition, we found a functionally critical region outside the transmembrane domain (TMD) core that is required for maximal protease activity. This region encompasses an additional 8-10 residues on the N-terminal side of the TMD core that precedes the first transmembrane segment and was not previously known to play a role in rhomboid function. These findings highlight the utility of the activity-based protein profiling approach for the characterization of rhomboid function.
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