(C-90) Structural Characterization of the Coiled-Coil Domain of the Liquid-Liquid Phase Separating Protein McdB

Liquid-liquid phase separation (LLPS) occurs when a homogenous solution experiences a change in conditions that causes two coexisting liquid phases.1,2 Generally, LLPS-forming proteins are either disordered or composed of large, coiled-coil domains.3  LLPS has recently been observed to have a crucial role in cellular organization and the formation of bacterial microcompartments. Dysregulation of LLPS has been associated with pathological protein aggregation linking it to neurodegenerative diseases, further emphasizing its importance in cellular functions.4 LLPS-forming polymers are a new potential avenue for developing drug delivery platforms.5  

The glutamine-rich McdB core domain (McdBq) was over-expressed in Arctic Express RIL DE3 cells and purified as previously described.6 For NMR spectroscopy, uniformly 15N-labeled McdBq was produced using M9 minimal media supplemented with 15N NH4Cl. For fluorescence and CD spectroscopy, samples were prepared with 3 μM McdB in 25 mM sodium phosphate (pH 5) or diethanolamine (pH 10) with 100 mM (low salt) or 500 mM (high salt) KCl. Tryptophan emission was measured using a Horiba Fluoromax 4c with 280 nm excitation, 290-440 nm emission, and 4 nm excitation and emission slit widths. Chemical denaturation was achieved using guanidinium hydrochloride or urea. NMR spectra were recorded on 300 μM McdBq in 50 mM sodium acetate, 25 mM Tris, 25 mM diethanolamine with 50 mM sodium chloride and 2 mM MgCl2 with 10% D2O. The NMR sample was prepared at pH 5 and titrated to higher pH by addition of 1 M NaOH.

Results, Conclusions, and Discussions:

The LLPS-inducing behavior of McdB has been shown to be both pH and salt dependent with LLPS propensity increasing with reduced pH and salt.6 Unfolding thermodynamics of McdBq were measured by quantifying the tryptophan fluorescence emission shifts in the presence of denaturant at high and low salt and at high and low pH (Figure 1). Redshift of the tryptophan emission indicates unfolding; loss of secondary structure at high denaturant was confirmed by CD (not shown). The spectral shift was interpreted as representative of the fraction of protein unfolded at each denaturant concentration. The Gibbs free energy of unfolding, indicated by the y-intercept in the right panel of Figure 2, was determined using a two-state model for unfolding. The data reveal a unique lack of cooperativity to McdBq unfolding under all conditions tested (Figure 2).

More detailed structural insight is revealed from the NMR spectra of McdBq at varying pH (Figure 3). 15N-HSQCs of McdB at pH 5.5, 6, and 8 show a distinct change in protein structure as pH increases. At low pH, well-defined peaks collapse near the center of the spectrum, indicating well-defined secondary structure but high variability in tertiary structure. As pH increases, the peaks spread out and become more resolved indicating a reproducible tertiary structure typical of a protein native state. The slight decrease in stability at pH 10 revealed by the fluorescence data indicates a small thermodynamic stabilization of the protein's molten globule state at low pH compared to the native state observed at high pH. This result may be due to increased protein-protein interactions between the LLPS-competent molten globules that both promote LLPS formation and provide stabilizing thermodynamic contributions. 

The noncooperative unfolding and small energy barrier between the native and molten globule states may also have important implications for McdB’s function in cellular environments. Carboxysome activity is known to follow intracellular pH changes, and the structural transition characterized by our data may reveal the mechanistic underpinnings of this behavior. Future work will provide more detailed structural characterization of the native state, thereby informing development of tunable, switchable LLPS-forming polymers.

Funding was provided by NSF 1941966 (AGV) and NSF 1942957 (NVN). HJT and KNP were supported by the National Institute of General Medical Sciences (T34GM136492). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

1. 
   

   Alberti, S., Gladfelter, A., & Mittag, T. (2019). Considerations and challenges in studying liquid-liquid phase separation and biomolecular condensates. Cell, 176(3), 419-434.

   
   


2. 
   

   Wang, B., Zhang, L., Dai, T., Qin, Z., Lu, H., Zhang, L., & Zhou, F. (2021). Liquid–liquid phase separation in human health and diseases. Sig Transduct Target Ther 6, 290. 

   
   


3. 
   

   Ramirez, D. A., Hough, L. E., & Shirts, M. R. (2023). Coiled-coil domains are sufficient to drive liquid-liquid phase separation of proteins in molecular models. bioRxiv.

   
   


4. 
   

   Babinchak, W. M., & Surewicz, W. K. (2020). Liquid–liquid phase separation and its mechanistic role in pathological protein aggregation. Journal of Molecular Biology, 432(7), 1910-1925.

   
   


5. 
   

   Baruch Leshem, A., Sloan-Dennison, S., Massarano, T., Ben-David, S., Graham, D., Faulds, K., Gottlieb, H. E., Chill, J. H., & Lampel, A. (2023). Biomolecular condensates formed by designer minimalistic peptides. Nature Communications, 14(1). 

   
   


6. 
   

   Basalla, J. L., Mak, C. A., Limcaoco, M. J., Hoang, Y., & Vecchiarelli, A. G. (2022). Dissecting the phase separation and oligomerization activities of the carboxysome positioning protein McdB. bioRxiv.