Putative Stabilizing Interior Mutations

General References

Conclusion

Hydrogen Bond Stabilizes a Beta-Barrel ProteinIntroduction

(Top) Amicyanin has one cross-barrel hydrogen bond between Trp45 and Tyr90. In the W45Y mutant this bond is lost. (Bottom) The W45Y amicyanin mutant has significantly decreased thermal stability as judged by the change in signal at 195 nm in the far UV circular dichroism spectra.

University of Central Florida, Burnett School of Biomedical Sciences, College of Medicine, Orlando, FLBrian A. Dow and Victor L. Davidson

Investigations Into Stabilizing Antibody-based Therapeutics

Amicyanin (PDB 2OV0) and human VL

(PDB 2Q2O) are aligned and shown in blue and pink, respectively.

Methods for improving the stability of these immunoglobulin domains can be gleaned from unexpected sources. An example of such an unexpected source is amicyanin. Amicyanin is a blue copper protein which mediates biological electron transfer via its metal center. Amicyanin has a striking structural similarity to the immunoglobulin VL. They consist of a beta-barrel structure with two beta sheets in a Greek key motif with no alpha helical secondary structure. When the beta sheets and a conserved beta loop are structurally aligned using either the RCSB jCE alignment algorithm, the resulting alignment RMSD is 3.1 Å. In amicyanin, it has been shown that a single interior cross-barrel hydrogen bond between Trp45 and Tyr90 that links the two beta-sheets significantly increases the thermal stability. When this bond was removed via a W45Y mutation, the structure of the protein remained unchanged, while the stability of the protein was significantly decreased. The VL does not contain any cross-barrel hydrogen bonds. Using the RCSB jCE structural alignment algorithm, an analogous location for the amicyanin cross-barrel hydrogen bond in the VL has been identified. I21W, I21Y, and I21W/T102Y mutations in the VL will be made in an attempt to create a cross-barrel hydrogen bond. This will help improve the stability and reduce the population of partially unfolded proteins, which lead to the formation of aggregates. By decreasing the aggregation kinetics, the shelf life of these new therapeutics as well as the ability to store them in the required high concentrations should be significantly improved.

An increasing number of recombinant human monoclonal antibody-based therapeutics are currently in clinical trials and being approved. Despite the increasing trend toward the use of therapeutic biologics, protein aggregation represents a major technical challenge in the manufacture, storage, and use of antibody based therapeutics. The aggregation characteristics of antibody based therapeutics is determined by the stability of the light and heavy chain variable domains. The heavy chain variable domain (VH) has been successfully engineered to exhibit more favorable biophysical properties. However, the intra-domain interactions responsible for the decreased stability and unfolding of the VL remain to be elucidated. Identification of these factors has proven difficult due to the high sequence diversity in the complementarity determining region (CDR), and the need to maintain antigen binding. This study proposes to study and stabilize the light chain variable domain of human immunoglobulins via its interior bond network.

Y90W/Y45

Trp45/Ile21Tyr90/Thr102

Far UV Circular Dichroism Spectra

200 210 220 230 240 250 260

-5

0

5

10

216 nm 232 nm

206 nm195 nm

220 nm

VLamicyanin

wavelength (nm)

[]x

10-3

(deg

cm

2 dm

ol-1

)

• Grow BL21 E. coli with recombinant VL gene

• Induce protein expression with IPTG for 20

hours at 30° C

• Prepare periplasm w/ lysozyme and

osmotic shock

• Supernatant purification using Ni-NTA resin

The KI O18/O8 germline VL and amicyanin both have similar secondary structure including, a beta-sheet secondary structure with a minimum at or near 220 nm. The VL has a second minimum at 232 nm, indicative of interactions of the aromatic residues with the sole tryptophan. Other spectral features of the VL include beta-turns and random coils, shown by a maximum and minimum at 205 nm and 200 nm, respectively. Amicyanin also has a maximum at 195 nm, which also represents the beta-sheet. These spectra show that both proteins are mostly beta-sheets with beta-turns. The beta-turns in the VL appear to be larger, and the random coils are the CDR. This spectrum shows that the expressed VL has the proper structure in solution.

Light Chain Variable Domain Expression

Structurally equivalent pairs in Amicyanin (blue) and the VL (pink). Mutagenesis of Ile21 to a hydrophobic residue whose side chain is capable of hydrogen bonding in the VL may lead to the formation of a stabilizing interior cross-barrel hydrogen bond with Thr102. Examples of such mutants include Ile21Trp and Ile21Tyr. Tryptophan is found in this location in amicyanin..

Amicyanin and Light Chain Variable Domain Sequence and Structural

Alignment Results

Sequence Identity 11%Sequence Similarity 22%Structure RMSD 2.9 Å

T7 PromoterLacOMauC Leader SequenceKI O8/O18 VL GeneHexa-His Tag

T7 TerminatorAmpicillin Resistance PromoterAmpicillin Resistance GeneLacI

Dow, B.A., et al., The sole tryptophan of amicyanin enhances its thermal stability but does not influence the electronic properties of the type 1 copper site. Arch Biochem Biophys, 2014. 550-551: p. 20-7.

Demarest, S.J. and S.M. Glaser, Antibody therapeutics, antibody engineering, and the merits of protein stability. Curr Opin Drug Discov Devel, 2008. 11(5): p. 675-87.

Lowe, D., et al., Aggregation, stability, and formulation of human antibody therapeutics. Adv Protein Chem Struct Biol, 2011. 84: p. 41-61.

Shindyalov, I.N. and P.E. Bourne, Protein structure alignment by incremental combinatorial extension (CE) of the optimal path. Protein Eng, 1998. 11(9): p. 739-47.

IgG antibody is the most common and simple antibody. It is

composed of two heavy chains (blue) and two light chains (red).

The VL is one half of the antigen binding fragment of human

immunoglobulins. Its CDRs are shown in green.

Amicyanin and VL share high structural alignment despite being from different protein families and

low sequence alignment.

VL Expression Plasmid VL Purification Process

VL Purification SDS-PAGE

Lane 1) MW Marker; Lane 2) Total Cell Extract; Lane 3) VL purified by Ni-NTA resin

Light Chain Variable Domain Structure

13kDa VL