The effects of a subclass of monoclonal antibodies (mAbs) on protein−protein interactions, formation of reversible oligomers (clusters), and viscosity (η) are not well understood at high concentrations. Herein, we quantify a short-range anisotropic attraction between the complementarity-determining region (CDR) and CH3 domains (K CDR-CH3 ) for vedolizumab IgG1, IgG2, or IgG4 subclasses by fitting small-angle X-ray scattering (SAXS) structure factor S eff (q) data with an extensive library of 12-bead coarse-grained (CG) molecular dynamics simulations. The K CDR-CH3 bead attraction strength was isolated from the strength of longrange electrostatic repulsion for the full mAb, which was determined from the theoretical net charge and a scaling parameter ψ to account for solvent accessibility and ion pairing. At low ionic strength (IS), the strongest short-range attraction (K CDR-CH3 ) and consequently the largest clusters and highest η were observed with IgG1, the subclass with the most positively charged CH3 domain. Furthermore, the trend in K CDR-CH3 with the subclass followed the electrostatic interaction energy between the CDR and CH3 regions calculated with the BioLuminate software using the 3D mAb structure and molecular interaction potentials. Whereas the equilibrium cluster size distributions and fractal dimensions were determined from fits of SAXS with the MD simulations, the degree of cluster rigidity under flow was estimated from the experimental η with a phenomenological model. For the systems with the largest clusters, especially IgG1, the inefficient packing of mAbs in the clusters played the largest role in increasing η, whereas for other systems, the relative contribution from stress produced by the clusters was more significant. The ability to relate η to shortrange attraction from SAXS measurements at high concentrations and to theoretical characterization of electrostatic patches on the 3D surface is not only of fundamental interest but also of practical value for mAb discovery, processing, formulation, and subcutaneous delivery.

We have solved the crystal structure of a segment of nonerythroid ␣-spectrin (␣II) consisting of the first 147 residues to a resolution of 2.3 Å . We find that the structure of this segment is generally similar to a corresponding segment from erythroid ␣-spectrin (␣I) but exhibits unique differences with functional significance. Specific features include the following: (i) an irregular and frayed first helix (Helix C); (ii) a helical conformation in the junction region connecting Helix C with the first structural domain (D1); (iii) a long A 1 B 1 loop in D1; and (iv) specific inter-helix hydrogen bonds/salt bridges that stabilize D1. Our findings suggest that the hydrogen bond networks contribute to structural domain stability, and thus rigidity, in ␣II, and the lack of such hydrogen bond networks in ␣I leads to flexibility in ␣I. We have previously shown the junction region connecting Helix C to D1 to be unstructured in ␣I (Park, S., Caffrey, M. S., Johnson, M. E., and Fung, L. W. (2003) J. Biol. Chem. 278, 21837-21844) and now find it to be helical in ␣II, an important difference for ␣-spectrin association with ␤-spectrin in forming tetramers. Homology modeling and molecular dynamics simulation studies of the structure of the tetramerization site, a triple helical bundle of partial domain helices, show that mutations in ␣-spectrin will affect Helix C structural flexibility and/or the junction region conformation and may alter the equilibrium between spectrin dimers and tetramers in cells. Mutations leading to reduced levels of functional tetramers in cells may potentially lead to abnormal neuronal functions.Spectrin isoforms are proteins associated with the cytoplasmic surface of plasma membranes of most cells. Spectrin associates with other cytoskeletal proteins, such as ankyrin and protein 4.1, to establish and maintain a diverse set of specialized plasma membrane domains (1). Nonerythroid, or brain, spectrin (spectrin II) exhibits high sequence homology with erythroid spectrin (spectrin I), despite their different cellular physiological functions (1). Because of their sequence homology, it is possible in theory to apply most of the molecular information obtained from the well studied spectrin I (both ␣I 3 and ␤I subunits) to the less studied spectrin II (␣II and ␤II) or from one domain to another domain (2). Yet, the two spectrin isoforms exhibit quite different functional properties as follows: (i) the ability of the spectrin I network to deform or the spectrin II network to remain rigid, and (ii) the ability ␣␤ heterodimers to associate to form functional (␣␤) 2 tetramers with much higher affinity in spectrin II than in spectrin I. Tetramer formation in nonerythroid spectrin is essential in the regulatory step for neuritogenesis (3). ␣II-spectrin has recently been reported to be essential for stabilizing nascent sodium channel clusters (4), assembling the mature node of Ranvier (4), and regulating endothelial cell-cell contacts (5).The C terminus of ␣I or ␣II and the N terminus of ␤I or ␤II associate to f...

Capreomycin is a potent ribosome-targeting antibiotic that is an essential component of current antituberculosis treatments, particularly in the case of multidrug-resistant Mycobacterium tuberculosis (Mtb). Optimal capreomycin binding and Mtb ribosome inhibition requires ribosomal RNA methylation in both ribosome subunits by TlyA (Rv1694), an enzyme with dual 2'-O-methytransferase and putative hemolytic activities. Despite the important role of TlyA in capreomycin sensitivity and identification of inactivating mutations in the corresponding Mtb gene tlyA, which cause resistance to capreomycin, our current structural and mechanistic understanding of TlyA action remains limited. Here, we present structural and functional analyses of Mtb TlyA interaction with its obligatory co-substrate for methyltransferase activity, S-adenosyl-l-methionine (SAM). Despite adopting a complete class I methyltransferase fold containing conserved SAM-binding and catalytic motifs, the isolated TlyA carboxyl-terminal domain exhibits no detectable affinity for SAM. Further analyses identify a tetrapeptide motif (RXWV) in the TlyA interdomain linker as indispensable for co-substrate binding. Our results also suggest that structural plasticity of the RXWV motif could contribute to TlyA domain interactions, as well as specific recognition of its two structurally distinct ribosomal RNA targets. Our findings thus reveal a novel motif requirement for SAM binding by TlyA and set the stage for future mechanistic studies of TlyA substrate recognition and modification that underpin Mtb sensitivity to capreomycin.

The global dissemination, potential activity in diverse species and broad resistance spectrum conferred by the aminoglycoside-resistance ribosomal RNA methyltransferases make them a significant potential new threat to the efficacy of aminoglycoside antibiotics in the treatment of serious bacterial infections. The N1 methylation of adenosine 1408 (m1A1408) confers resistance to structurally diverse aminoglycosides, including kanamycin, neomycin and apramycin. The limited analyses to date of the enzymes responsible have identified common features but also potential differences in their molecular details of action. Therefore, with the goal of expanding the known 16S rRNA (m1A1408) methyltransferase family as a platform for developing a more complete mechanistic understanding, we report here the cloning, expression and functional analyses of four hypothetical aminoglycoside-resistance rRNA methyltransferases from recent genome sequences of diverse bacterial species. Each of the genes produced a soluble, folded protein with a secondary structure, as determined from circular dichroism (CD) spectra, consistent with enzymes for which high-resolution structures are available. For each enzyme, antibiotic minimum inhibitory concentration (MIC) assays revealed a resistance spectrum characteristic of the known 16S rRNA (m1A1408) methyltransferases and the modified nucleotide was confirmed by reverse transcription as A1408. In common with other family members, higher binding affinity for the methylation reaction by-product S-adenosylhomocysteine (SAH) than the cosubstrate S-adenosyl-L-methionine (SAM) was observed for three methyltransferases, while one unexpectedly showed no measurable affinity for SAH. Collectively, these results confirm each hypothetical enzyme is a functional 16S rRNA (m1A1408) methyltransferase but also point to further potential mechanistic variation within this enzyme family.

Significance The bacterial ribosome is an important target for antibiotics used to treat infection. However, resistance to these essential drugs can arise through changes in ribosomal RNA (rRNA) modification patterns through the action of intrinsic or acquired rRNA methyltransferase enzymes. How these antibiotic resistance-associated enzymes recognize their ribosomal targets for site-specific modification is currently not well defined. Here, we uncover the molecular basis for large ribosomal (50S) subunit substrate recognition and modification by the Mycobacterium tuberculosis methyltransferase TlyA, necessary for optimal activity of the antitubercular drug capreomycin. From this work, recognition of complex rRNA structures distant from the site of modification and “flipping” of the target nucleotide base both emerge as general themes in ribosome recognition for bacterial rRNA modifying enzymes.