Using Engineered Conditional Stability to OptimizeDimerization of a Synthetic Ligand Binding Protein

Colton McDavid2, Benjamin W. Jester1,2, Stanley Fields1,2,3 1Howard Hughes Medical Institute, University of Washington, Seattle, WA. 2Department of Genome Sciences, University of Washington, Seattle, WA. 3Department of Medicine, University of Washington, Seattle, WA.

PAIRING DIG AND PRO DIMERSSeveral mutations to the ligand-binding domain of DIG 10.3 changed the ligand-binding specificity from digoxigenin to progesterone (PRO). We paired five of these progesterone variants with Dig 10.3 and with each other using luciferase expression as a quantitative alternative to HIS3 expression.

CONCLUSION AND FUTURE DIRECTIONS

ACKNOWLEDGEMENTS

Utilizing directed evolution, we identified several converging mutations that appear to have a stabilizing influence on DIG 10.3’s dimer interface. We are continuing to test the effects of each mutation on DIG 10.3. Our goal is to identify mutations that increase the affinity of DIG to form hetero-dimers over non functional homo-dimers and apply these mutations to our DIG/PRO dimer. Such mutations provide a modular system with a variety of potential downstream applications including biosensors and orthogonal gene circuits.

Special thanks to Ben Jester and Stan Fields for the opportunity to engage in undergraduate research and to Christine Tinberg and David Baker for DIG 10.3.The UW Undergraduate Research Program for the experience of working in a lab and HHMI for funding my work.

Of continuing importance in the field of synthetic biology is the development of tools to aid in understanding protein-protein interfaces and inter-protein interactions. To this end, we developed a method utilizing directed evolution to optimize the dimerization of DIG 10.3 (DIG), a synthetic protein engineered to bind the small molecule digoxigenin. We exploited the inherent instability of DIG to isolate variants that are dependent on the presence of digoxigenin for stability and are rapidly degraded in its absence. We then identified pairs of these conditionally stable variants that preferentially form hetero-dimers over homo-dimers and used them to identify mutations to the protein-protein interface that optimize dimerization of DIG in the presence of ligand.

ABSTRACT

GENERAL METHODOur system consists of a DNA binding domain, Gal4, and a transcriptional activation domain, VP16, which when brought into proximity recruit RNA polymerase to the HIS3 reporter gene. One copy of Dig 10.3 is fused to Gal4 and one to VP16 such that dimerization of DIG 10.3 in the presence of digoxigenin leads to transcription. Because both Gal4 and VP16 are necessary for transactivation, DIG hetero-dimers activate transcription but homo-dimers are nonfunctional.

IDENTIFYING THE BEST DIG 10.3 HETERODIMERSWe paired several dimer interface mutants of DIG 10.3 and assessed their dimer stability by comparing growth in minimal media lacking histidine and containing digoxigenin and 3AT, a competitive inhibitor of the HIS3 gene product. The ability of S. cerevisiae to grow depends on the successful dimerization of DIG.

Fig 1. DIG 10.3 dimerizes in the presence of digoxigenin to activate transcription of the reporter gene. Without digoxigenin, DIG 10.3 is rapidly degraded.

Fig 3. Relative luminescence data following the addition of D-Luciferin to DIG-containing yeast allowed us to determine the best hetero-dimer pairs. A DMSO control was used at <1% total concentration because both digoxigenin and progesterone were dissolved in DMSO.

Fig 5. Several converging mutations came out of our selection and the wild type sequence was enriched by about 5-fold, which suggests that the selection successfully enriched for stable dimers.

1. Tinberg, C.E., et al. Nature 501, 212-216 (2013).REFERENCES

Gal-DIGDesignation

DIG-VP16 Designation Mutation(s)

GDWT DVWT NONE

GD1 DV1 E83V

GD2 DV2 E83V, P127S

GD3 DV3 V104A

GD4 DV4 Y36H

GD5 DV5 I81F

p415cyc p416cyc EMPTY

Gal-PRODesignation

PRO-VP16 Designation Mutation(s)

GPWT PVWT NONE

GP1 PV1 Y101F

GP2 PV2 Y34F, Y101F, Y99F (3F)

GP3 PV3 3F, K96M, L14Q, F19Y, V124E

GP4 PV4 3F, K96E, V104A

GP5 PV5 3F, H9R, E15G, I64F, A92T

p415cyc p416cyc EMPTY

Fig 4. DIG1 (shown on the right) contains the mutation E83V.Mutants of DIG 10.3 (shown on the left) were selected to dimerize with DIG1. Both proteins are shown binding digoxigenin.

Fig 7. Mutations produced by our selection favor dimerization with DIG 1 over DIG 4. Expression of luciferase increased further by combining our mutants with DIG 1b, which contains the mutation E83R.

37/DV1

37/DV4

37/DV1b

59/DV1

59/DV4

59/DV1b

37,59/DV1

37,59/DV4

37,59/DV1b

37,59,95/DV1

37,59,95/DV4

37,59,95/DV1b

GD/DV

GD/DV1

GD/DV4

GD/DV1b

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Luciferase Expression of Selection Mutants

DMSO5 Dig50 Dig

Fig 2. Our initial DIG variants (their specific mutations described on the left) were assayed for growth on plates lacking histidine and containing 10 or 100µM digoxigenin and 1 µM 3AT.

1 mM 3AT x 100µM digoxigenin 100 µM digoxigenin

1mM 3AT x 10 µM digoxigenin 10 µM digoxigenin

GD5 x DV1

GD3 x DV1

GD2 x DV1

GD1 x DV1

GD x DV4

GD x DV1

P415cyc x DV1

0

2

4

6

8Convergent Mutations to Dig 10.3

P37S P37L P37Q P59L T43S L25M R123S I6T G95D

Freq

uenc

y

IDENTIFYING CONVERGENT MUTATIONSUsing the most stable DIG 10.3 heterodimer, GD/DV1, we mutagenized the DIG domain of the Gal-Dig 10.3 fusion using error-prone PCR. We then plated our mutant library on media lacking histidine and containing digoxigenin. Of 98 sequences representing the largest colonies on the plates, we identified several recurring mutations to the same subset of residues, suggesting that our selection succeeded in enriching for stabilizing mutations.

DIG 10.3 domain fused to Gal4 DIG1 (E83V) fused to VP16

GD/DV1 PG2/PV2 PG2/DV10

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Luciferase Expression of Best Dimer Pairs

DMSO100µM Dig10µM Dig10µM Pro1µM Pro100µM Dig/ 10µM Pro100µM Dig/1µM Pro10µM Dig/1µM Pro

TESTING SELECTION MUTANTSWe used site-directed mutagenesis to generate some of the mutants from the selection clone them into plasmids. In order to assess the stabilizing effects of each mutation, we gathered both quantitative and qualitative data using both the HIS3 and luciferase reporters.

Fig 6. Mutants produced by our selection grow as well as, if not better than GDWT when paired with DV1, suggesting that the converging mutations that came out of the selection are stabilizing.

1 mM 3AT x 1 µM digoxigenin 1 mM 3AT x 10 µM digoxigenin