Structure-Based Prediction of Asparagine and Aspartate Degradation Sites in Antibody Variable Regions

Monoclonal antibodies (mAbs) and proteins containing antibody domains are the most prevalent class of biotherapeutics in diverse indication areas. Today, established techniques such as immunization or phage display allow for an efficient generation of new mAbs. Besides functional properties, the stability of future therapeutic mAbs is a key selection criterion which is essential for the development of a drug candidate into a marketed product. Therapeutic proteins may degrade via asparagine (Asn) deamidation and aspartate (Asp) isomerization, but the factors responsible for such degradation remain poorly understood. We studied the structural properties of a large, uniform dataset of Asn and Asp residues in the variable domains of antibodies. Their structural parameters were correlated with the degradation propensities measured by mass spectrometry. We show that degradation hotspots can be characterized by their conformational flexibility, the size of the C-terminally flanking amino acid residue, and secondary structural parameters. From these results we derive an accurate in silico prediction method for the degradation propensity of both Asn and Asp residues in the complementarity-determining regions (CDRs) of mAbs

Biochemical and Molecular Characterization of the Biosynthesis of Glutamine and Glutamate, Two Major Compatible Solutes in the Moderately Halophilic Bacterium Halobacillus halophilus

The moderately halophilic, chloride-dependent bacterium Halobacillus halophilus produces glutamate and glutamine as main compatible solutes at external salinities of 1.0 to 1.5 M NaCl. The routes for the biosynthesis of these solutes and their regulation were examined. The genome contains two genes potentially encoding glutamate dehydrogenases and two genes for the small subunit of a glutamate synthase, but only one gene for the large subunit. However, the expression of these genes was not salt dependent, nor were the corresponding enzymatic activities detectable in cell extracts of cells grown at different salinities. In contrast, glutamine synthetase activity was readily detectable in H. halophilus. Induction of glutamine synthetase activity was strictly salt dependent and reached a maximum at 3.0 M NaCl; chloride stimulated the production of active enzyme by about 300%. Two potential genes encoding a glutamine synthetase, glnA1 and glnA2, were identified. The expression of glnA2 but not of glnA1 was increased up to fourfold in cells adapted to high salt, indicating that GlnA2 is the glutamine synthetase involved in the synthesis of the solutes glutamate and glutamine. Furthermore, expression of glnA2 was stimulated twofold by the presence of chloride ions. Chloride exerted an even more pronounced effect on the enzymatic activity of preformed enzyme: in the absence of chloride in the assay buffer, glutamine synthetase activity was decreased by as much as 90%. These data demonstrate for the first time a regulatory role of a component of common salt, chloride, in the biosynthesis of compatible solutes

Human mitochondrial transcription factor A induces a U-turn structure in the light strand promoter

Human mitochondrial transcription factor A, TFAM, is essential for mitochondrial DNA packaging and maintenance and also has a crucial role in transcription. Crystallographic analysis of TFAM in complex with an oligonucleotide containing the mitochondrial light strand promoter (LSP) revealed two high-mobility group (HMG) protein domains that, through different DNA recognition properties, intercalate residues at two inverted DNA motifs. This induced an overall DNA bend of ~180°, stabilized by the interdomain linker. This U-turn allows the TFAM C-terminal tail, which recruits the transcription machinery, to approach the initiation site, despite contacting a distant DNA sequence. We also ascertained that structured protein regions contacting DNA in the crystal were highly flexible in solution in the absence of DNA. Our data suggest that TFAM bends LSP to create an optimal DNA arrangement for transcriptional initiation while facilitating DNA compaction elsewhere in the genome.This study was supported by the Ministerio de Ciencia e Innovación (grants BFU2006-09593 to M.S., BFU2009-07134 to M.S., BFU2008-02372 to M.C., CSD2006-00023), Generalitat de Catalunya (SGR2009-1366 to M.S., SGR2009-1309 to M.C., SGR2009-1352 to P.B.), the European Union (FP7-HEALTH-2010-261460 to M.S., FP7-BioNMR-2010-261863 to P.B.), and Instituto de Salud Carlos III-FIS-PI 10/00662. The Centro de Investigación Biomédica en Red de Enfermedades Raras is an initiative of the Instituto de Salud Carlos III. A.R.-C., J.F.S., N.J.-M. and P.F.-M. hold or held fellowships from Consejo Superior de Investigaciones Científicas, MICINN and Cusanswerk-Bischöfliche Studienförderung.Peer Reviewe

Experimental Asn and Asp hotspot collection.

<p>*only Asp as deamidation species.</p>‡<p>excluded from hotspot data set because of interaction with a CDR glycosylation site which is not represented by the homology models.</p>#<p>proof of modification site impossible with available methods (tryptic peptide, AspN peptide, CID fragmentation, HCD fragmentation), thus excluded from the hotspot data set.</p><p>Main modifications are written in bold. iD = isomerization, suc = succinimide, dea = deamidation, n.a.: not assessed.</p

Parameters characterizing Asn and Asp residues in a structural environment outlined at an exemplary Asp residue.

<p>Parameters describing the carboxyl/amino group leaving tendency, the transition state accessibility, the N_n+1 nucleophilicity, and the structural environment are depicted in pink, light blue, purple, and dark blue, respectively. Parameter names are used as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100736#pone.0100736.s003" target="_blank">Table S1</a>.</p

Asparagine and Aspartate degradation pathways.

<p>Deamidation of asparagine or dehydration of aspartic acid occurs by nucleophilic attack of the α-amino group of the C-terminally flanking amino acid. This leads to formation of a metastable succinimide (cyclic imide) intermediate, which hydrolyzes to a mixture of aspartyl and iso-aspartyl linkages. Alternatively, nucleophilic attack by the backbone carbonyl oxygen results in a cyclic isoimide intermediate, yielding only aspartyl residues after hydrolysis independent of the point of attack of the incoming water molecule. Asparagine residues can deamidate to Asp by direct water-assisted hydrolysis. Standard amino acids (Asn, Asp) are outlined with black boxes.</p

Final Aspartate (A) and asparagine (B) decision trees.

<p>The outline of nodes and leaves is colored by the weighted majority of the class that is present (red: hotspots, green: non-hotspots). Filling levels of the bars on the right hand side of each node/leaf refer to the fraction of the data set. The fraction of each class at a node/leaf is shown by the colored fraction of the circle. The number of members of each node/leaf is indicated above.</p