Physical Models of Allostery: Allosteric Regulation in Capsid Assembly

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1 Physical Models of Allostery: Allosteric Regulation in Capsid Assembly QCB Journal Club Prof. Sima Setayeshgar JB Holmes Nov. 2, 2017

2 Mechanisms of Allosteric Regulation From R.A. Laskowski, FEBS Letters, 2009 Allosteric activators and inhibitors (some common mechanisms)

3 A Review on Cooperative Binding Enzyme catalysis can be divided into steps and more than one substrate can be bound E + S!! ES!! E + P V 0 = V max[s] K m + [S] Out[6]= V0 [S]

4 A Review on Cooperative Binding E + S!! ES + S!!! ES 2 + S!!!... Positive cooperativity : Substrate binding increases affinity for more substrate Negative cooperativity: Substrate binding decreases affinity for more substrate Non-cooperative binding: Substrate binding does not affect binding affinity for subsequent substrate

5 An Example Hemoglobin Hb" Hb(O2)4 Positive Myoglobin Mb MbO2 Non-cooperative V 0 = V max [S] K m + [S] From Interactive Biochemistry, Wiley, 2002

6 The Concerted or Symmetry or MWC Model 1. Cooperative proteins are composed of several identical units that occupy equivalent positions within the protein 2. The binding sites within each protein are equivalent. 3. The protein has two conformational states, usually denoted by R and T, which differ in their ability to bind ligands. From J. P. Changeux, Annu. Rev. Biophys. (2012) Hemoglobin

7 The Sequential (KNF) Model Hemoglobin 1. Intermediate exist between T and R states 2. Change in conformation after a binding event 3. Cooperativity is easily seen as an increase in the affinity to adjacent subunits

8 More on the MWC Model of Allostery From S. Marzen, J. Mol. Biol., 2009 From S. Marzen, J. Mol. Biol., 2009

9 Concerted (MWC) and Sequential (KNF) Models From Bialek and Setayeshgar, PRL (2008)

10 The Virus Life Cycle (HBV) Capsid components must be organized at the right place and time to ensure assembly of infectious particles Theoretically we might expect an allosteric mechanism to control/ enhance assembly in the cell Solution studies suggest that the assembly-inactive state is more accurately an ensemble of conformations. Here we will focus on allostery at the level of protein-protein interactions in the selfassembled virus capsid From qiagen.com, 2017

11 Assembly of Icosahedral Shells From C. Packinathan, Journal of Virology 2010 Out[211]= From Zlotnick, Proc. Natl. Acad. Sci. U. S. A Competing thermodynamic and kinetic constraints Strategies to control capsid assembly (self-regulation of the assembly kinetics) Assembly of rigid subunits into ordered structures requires weak, reversible interactions (compared to the thermal energy)

12 Allosteric Models (With and Without Induced Fit) K A = e g A/apple B T From M. Hagen, Journal of Physical Chemistry B, 2016

13 Computational and Master Equation Models of Assembly Brownian Dynamics (course grained) Simulations Rate and Dissipation Equations dc 1 dt = k 1c k 2 c 2 + dc n dt = k n 1c 1 c n 1 k n c 1 c n kn c n + k n+1 c n+1, n =2...N NX n=2 k n c n c 1 + k n c n free subunit From M. Hagen, Journal of Physical Chemistry B, 2016

14 Results: No Allostery BD and Master equation models with no allostery as a function of time From M. Hagen, Journal of Physical Chemistry B, 2016

15 Results: No Allostery From M. Hagen, Journal of Physical Chemistry B, 2016 Complete capsid and intermediates as a function of binding energies when no allostery is considered

16 Results: Allosteric Models From M. Hagen, Journal of Physical Chemistry B, 2016 Assembly robustness depends on the strength of allostery. Plotted are two different values of ga, compared with no allostery results from BD simulations

17 Results: Induced Fit ga = 4 (sufficient bias on the inactive state) Productive assembly occurs over a greater range of binding energies From M. Hagen, Journal of Physical Chemistry B, 2016

18 Summary and Conclusions The analysis focused on capsid size and parameters consistent with experiments on HBV (SEC and Light scattering experiments) Placing a sufficient bias towards the inactive conformation allows productive assembly at very high concentrations or binding affinities over a broader range for the parameters Increasing ga, can be thought of as changing solution parameters such as salt concentration and ph and has an affect on assembly robustness which can be tuned in the simulations The inactive and active conformations can also be influenced by reactions with virus components, such as RNA or DNA binding, but such ensembles with significant structural diversity were not considered here. However, understanding allosteric mechanisms and how they cooperate to control the time, place, and rate of assembly will lead to better understanding of the virus life cycle and could lead to new strategies for synthetic assembly systems

19 Discussion 1.One of the keys to the MWC model is that when a ligand binds, it shi:s the conforma=on of all subunits into the ac=ve state. Another model suggests that when a ligand binds, the conforma=on of other subunits does not necessarily have to change. Is one of these models more accurate, or is the appropriate model a case by case situa=on? When would it be biologically beneficial to have a macromolecule func=on as an MWC model vs. not? 2.This paper models a simple viral capsid consis=ng of only its one coat protein, so it fits very well with the MWC model. Does this model break down if you complicate the capsid with something like a scaffold protein or a portal protein that makes the capsid a heterooligomer of sorts? Is it possible for other components of the capsid to also play into an induced fit model and convert a subunit from an inac=ve to an ac=ve state? 3.What would be a good experimental set up to observe allosteric capsid assembly in vitro? Specifically what methods would be best for measuring this (FRET, ITC, SPR, etc.) and why? 4.Taking the ideas from this paper an applying them: as far as I m aware, not a lot of an=viral strategies that target the assembly of viruses. Would designing a small molecule that traps a subunit into an ac=ve form leading to kine=cally trapped incorrectly assembled capsids be worth pursing? What would be some poten=al problems of doing this?