Sara Remmler, Biochemistry
Understanding Membrane Fusion and Function of the SARS-CoV-2 Spike protein
Project Advisor: Amy Camp
The coronavirus has affected us all unlike any other pandemic or outbreak. The highly infectious SARS-CoV-2 virus has infected over 470 million people and has taken the lives of over 6.1 million people worldwide1.What do we know exactly about the SARS- CoV-2 virus and spike protein, and how much is there still left to discover? These were the questions Daniel Birtles and Jinwoo Lee attempted to answer in their 2021 paper Identifying Distinct Structural Features of the SARS-CoV-2 Spike Protein Fusion Domain Essential for Membrane Interaction2 .
The SARS-Co-V-2 spike protein, which allows the virus to enter and wreak havoc in the host cell, has been highly researched, but the process of membrane fusion, facilitated by the fusion domain (FD) remains unclear. In this study, a forty amino acid construct was expressed and purified to replicate a FD which consists of two different regions, the fusion peptide (FP) and the fusion loop (FL). Nuclear Magnetic Resonance (NMR), Circular Dichroism (CD) Spectroscopy, Paramagnetic Relaxation Enhancement (PRE), and Cryo-Electron Microscopy (Cryo-EM), revealed that when the replicated FD interacted with the mimic host membrane the FP, originally in a random coil, underwent a large conformational structural change to form a helix turn helix motif and thus, embed itself into the host membrane. In this presentation I will explain the bipartite fusion platform model created by Birtles and Lee, explain key challenges in this study, and what makes the SARS-CoV-2 virus different from other members of the coronavirus family, Influenza, HIV, and EBOV.
1 WHO coronavirus (COVID-19) dashboard. https://covid19.who.int/ (accessed Mar 23, 2022).
2 Birtles, D., and Lee, J. (2021) Identifying Distinct Structural Features of the SARS-CoV-2 Spike Protein
Fusion Domain Essential for Membrane Interaction, Biochemistry 60, 2978-2986.
Ray Stieber, Biological Sciences
Exploring the Interaction Between the Bacillus subtilis Protease ClpCP and a Novel Sporulation-Specific Adaptor Protein
Project Advisor: Dr. Amy Hitchcock Camp
To properly function and survive, cells across all domains of life need to be able to target and degrade specific proteins in order to accommodate changing, unstable environmental conditions. Proteases, or protein complexes that degrade other proteins, are commonly used by cells for these purposes. Many bacterial proteases, such as the AAA+-ATPase chaperone-protease ClpCP, require adaptor proteins for both activation and specificity in which proteins are recruited for degradation. A novel adaptor protein, MicA (metabolic inhibitor candidate A), has been identified by previous members of the Camp Lab and is hypothesized to interact with the aforementioned protease chaperone ClpC to degrade key metabolic enzymes during spore formation by the bacterium Bacillus subtilis. My project aims to further explore the interactions of MicA and ClpC as an adaptor-protease complex. The co-crystal structure of the MicA-ClpC complex developed by collaborators revealed three sites of electrostatic interactions between the novel adaptor and known protease, one of which being the MicA E53 - ClpC H79 site. Through my project, I am investigating the importance the aforementioned site has on the overall interactions between MicA and ClpC. Various point mutations of the site were tested through the use of an E. coli bacterial two-hybrid assay to demonstrate how MicA E53 and ClpC H79 affect the interactions of MicA and ClpC at large. The results and implications of these experiments will be explored and discussed in my presentation.
Imaan Moin, Biochemistry
Investigating the Interaction Between a Novel Adaptor Protein and the Bacillus subtilis Protease ClpCP
Project advisor: Dr. Amy Hitchcock Camp
The building blocks of life – carbohydrates, lipids, nucleic acids, and proteins – are constantly being built, broken down, and reshuffled into new macromolecules. When it comes to the life cycle of a protein, proteases (which are also a type of protein) break them down. In the bacteria Bacillus subtilis, the AAA+-ATPase chaperone-protease ClpCP is known to degrade proteins, and now a previously uncharacterized protein – MicA – has been hypothesized to function as a novel adaptor protein for ClpCP that possibly leads to the targeted degradation of metabolic enzymes in the developing B. subtilis spore during the process of sporulation. This working model was developed by the Camp Lab from prior members’ genetic and biochemical research, and current studies in the lab focus on the interaction between MicA and ClpC. Collaborators created a co- crystal structure of the two proteins, revealing three sites of electrostatic interactions between MicA and ClpC. In this project, I have been studying the importance of the electrostatic interactions at the ClpC K12 - MicA E161 site using previous lab members’ point-mutated variants. The interactions of the ClpC and MicA mutants were assessed using an E. coli based two-hybrid assay, revealing the importance of both the E161 and K12 sites to facilitate a MicA-ClpC interaction. These findings lead to a greater understanding of how adaptor proteins like MicA can interact with protein degradation machines like ClpCP and, in turn, how this interaction drives metabolic protein degradation during B. subtilis spore formation.
Gabi Davis, Biochemistry
Comparing the “MecA”-nisms of Two Adaptor Proteins Binding to the Bacillus subtilis Protease ClpCP
Project Advisor: Dr. Amy Hitchcock Camp
The aggregation of misfolded proteins in a cell can have deadly consequences, known as the primary cause for diseases like Alzheimer's and Parkinson’s Disease. Putting bacteria into perspective, this type of damage is crucial to recognize because without it damage can trigger the cell to upregulate more damaging proteins. In cases like these, it is crucial that the cell has a quality control system, such as protein degradation, in which enzymes are capable of breaking down individual damaged proteins before the problem starts. AAA+ protease-chaperone complexes, like ClpCP are capable of degrading these substrates, with the help of adaptor proteins who are responsible for recruiting these proteins marked for destruction. Previous members of the Camp Lab have identified a novel adaptor group collaborating with the complex ClpC; MicA, important for its role in the formation of the forespore during dormancy in Bacillus Subtilis. While MicA has not been thoroughly proven to work as an adaptor for ClpC, MecA, a heavily reviewed protein with no recorded connection to sporulation serves as both a model and an integral piece in understanding MicA. What remains unknown concerns MicA’s amino acid specificity and its collateral effects on MecA. Previous work has generated co-crystalline structures regarding the interactions between ClpC and both MicA and MecA and based on this data, my project revolved around the hypothesis that these amino acids should be specifically required for interactions with MicA, but could have indirect consequences to MecA as well. The execution of this experiment required the creation of mutant ClpC proteins in Escherichia coli which lack the sites K85, H79, and K12 producing our main data via a two-hybrid assay in the presentation to come.
Sandra Obwar, Biological Sciences
Characterizing the in vivo Interaction of the Bacillus Subtilis ClpC Chaperone with a Novel Sporulation Adaptor Protein
Project Advisor: Dr. Amy Hitchcock Camp
Some bacteria respond to stressful environmental conditions such as nutrient limitation by forming spores to protect themselves against ecological degrading agents. This process is known as sporulation and the spores formed are the most dormant form of bacteria, capable of surviving extreme conditions. The rod-shaped gram positive bacterium Bacillus subtilis that is mainly found in soil, is best known for producing spores. Although B. subtilis is widely studied, the mechanisms that lead to metabolic shutdown are still poorly understood. Previous members of the Camp lab have utilized both biochemical and genetic approaches to come up with a working model in which an uncharacterized protein “MicA” is hypothesized to function as a novel adaptor for the AAA+-ATPase chaperone protease ClpCP. The working model hypothesizes that MicA recruits metabolic enzymes to be degraded by ClpCP in the developing spore which eventually lead to metabolic shutdown. Through the help of our collaborators, a co-crystal structure was generated that revealed three sites of electrostatic interactions between MicA and ClpC, one of which is the YjbA glutamic acid (E) 53 - ClpC histidine (H) 79. In my project, I have hypothesized that ClpC H79 is necessary for in vivo interaction of these two proteins. To test this hypothesis, I created mutant B. subtilis strains harboring point mutations at the clpC H79 codon, switching it to a codon for alanine (A) or glutamic acid (E). I confirmed that these clpCH79A and clpCH79Emutations, as expected, do not cause a severe loss of ClpC function and am currently assessing them for in vivo interaction with MicA. Data from these experiments will be presented, and their implications for my hypothesis will be discussed.