Protein re-surfacing of E. coli L-Asparaginase to evade pre-existing anti-drug antibodies and hypersensitivity responses.

The optimal use of many biotherapeutics is restricted by Anti-drug antibodies (ADAs) and hypersensitivity responses which can affect potency and ability to administer a treatment. Here we demonstrate that Re-surfacing can be utilized as a generalizable approach to engineer proteins with extensive surface residue modifications in order to avoid binding by pre-existing ADAs. This technique was applied to E. coli Asparaginase (ASN) to produce functional mutants with up to 58 substitutions resulting in direct modification of 35% of surface residues. Re-surfaced ASNs exhibited significantly reduced binding to murine, rabbit and human polyclonal ADAs, with a negative correlation observed between binding and mutational distance from the native protein. Reductions in ADA binding correlated with diminished hypersensitivity responses in an in vivo mouse model. By using computational design approaches to traverse extended distances in mutational space while maintaining function, protein Re-surfacing may provide a means to generate novel or second line therapies for life-saving drugs with limited therapeutic alternatives.

Evolutionary pathways of repeat protein topology in bacterial outer membrane proteins.

Outer membrane proteins (OMPs) are the proteins in the surface of Gram-negative bacteria. These proteins have diverse functions but a single topology: the ß-barrel. Sequence analysis has suggested that this common fold is a ß-hairpin repeat protein, and that amplification of the ß-hairpin has resulted in 8-26-stranded barrels. Using an integrated approach that combines sequence and structural analyses, we find events in which non-amplification diversification also increases barrel strand number. Our network-based analysis reveals strand-number-based evolutionary pathways, including one that progresses from a primordial 8-stranded barrel to 16-strands and further, to 18-strands. Among these pathways are mechanisms of strand number accretion without domain duplication, like a loop-to-hairpin transition. These mechanisms illustrate perpetuation of repeat protein topology without genetic duplication, likely induced by the hydrophobic membrane. Finally, we find that the evolutionary trace is particularly prominent in the C-terminal half of OMPs, implicating this region in the nucleation of OMP folding.

Efflux Pumps Represent Possible Evolutionary Convergence onto the ß-Barrel Fold.

There are around 100 varieties of outer membrane proteins in each Gram-negative bacteria. All of these proteins have the same fold-an up-down ß-barrel. It has been suggested that all membrane ß-barrels excluding lysins are homologous. Here we suggest that ß-barrels of efflux pumps have converged on this fold as well. By grouping structurally solved outer membrane ß-barrels (OMBBs) by sequence we find that the membrane environment may have led to convergent evolution of the barrel fold. Specifically, the lack of sequence linkage to other barrels coupled with distinctive structural differences, such as differences in strand tilt and barrel radius, suggest that the outer membrane factor of efflux pumps evolutionarily converged on the barrel. Rather than being related to other OMBBs, sequence and structural similarity in the periplasmic region of the outer membrane factor of efflux pumps suggests an evolutionary link to the periplasmic subunit of the same pump complex.

Tight Turns of Outer Membrane Proteins: An Analysis of Sequence, Structure, and Hydrogen Bonding.

As a structural class, tight turns can control molecular recognition, enzymatic activity, and nucleation of folding. They have been extensively characterized in soluble proteins but have not been characterized in outer membrane proteins (OMPs), where they also support critical functions. We clustered the 4 to 6 residue tight turns of 110 OMPs to characterize the phi/psi angles, sequence, and hydrogen bonding of these structures. We find significant differences between reports of soluble protein tight turns and OMP tight turns. Since OMP strands are less twisted than soluble strands, they favor different turn structures types. Moreover, the membrane localization of OMPs yields different sequence hallmarks for their tight turns relative to soluble protein turns. We also characterize the differences in phi/psi angles, sequence, and hydrogen bonding between OMP extracellular loops and OMP periplasmic turns. As previously noted, the extracellular loops tend to be much longer than the periplasmic turns. We find that this difference in length is due to the broader distribution of lengths of the extracellular loops not a large difference in the median length. Extracellular loops also tend to have more charged residues as predicted by the charge-out rule. Finally, in all OMP tight turns, hydrogen bonding between the side chain and backbone 2 to 4 residues away from that side chain plays an important role. These bonds preferentially use an Asp, Asn, Ser, or Thr residue in a beta or pro phi/psi conformation. We anticipate that this study will be applicable to future design and structure prediction of OMPs.