Final Report Summary - INFEMEC (Nanomechanics of proteins involved in viral and bacterial infections)
Microbial infections are one of the main causes of death worldwide. Infections by viruses and bacteria cause diseases such as AIDS, pneumonia, tuberculosis, cholera or miningitis which are some of the most prominent infections particularly detrimental in underdeveloped countries. In addition, bacterial infections are now a global concern due to the increasing virulence and resistance to antibiotics of many bacterial strains. This is a problem not only affecting the developing world but also the so-called “First World”, where thousands of people die every year due to bacterial infections especially in medical settings . Therefore, it is recognized that viral and bacterial infections are a global problem that requires new research for the development of novel treatments. This new research best involves multidisciplinary and innovative techniques such as nanotechnology and biotechnology since traditional cell and molecular biology have proven to be insufficient to fully control infections.
Bacteria and viruses infect organisms by using proteins that attach to molecules in the surface of the host. HIV/AIDS affects over 30 million people worldwide. HIV-1 uses its envelope glycoprotein gp120 to attach to CD4 in the surface of T cell. Similarly, the bacterium E. coli uses an array of proteins called pilus for establishing mechanical anchoring to tissues. These proteins withstand mechanical forces that go from few to hundreds of picoNewtons. The effect of these forces in the structure and chemistry of the proteins is not understood but it may have implications in the infection process. In this project, we have investigated the role of mechanical forces in the structure and chemistry of the microbial attachment proteins as well as the infection process. We have used an array of techniques to study the nanomechanics of viral and bacterial infections progressively from single molecules to cells. We have established new knowledge of the molecular aspects that drive the mechanical interaction of microbes with their targets. First, we have used atomic force spectroscopy to explore the effect of mechanical forces in microbial attachment proteins, human CD4 and E. coli pilus. This technique allows monitoring chemical reactions under force such as the reduction of disulfide bonds or the binding of peptides, small molecules and antibodies, processes which are known to be implicated in microbial infections and that may have a mechanical origin. Second, we have used bioinformatics to search for molecules that alter the mechanical properties of these anchoring proteins and that can potentially be used to prevent infections. Viral and bacterial infections have been widely studied but there is not much information in the context of mechanical interactions. We believe that our project is providing a new understanding about infectious diseases.
Under this grant we have made substantial progress to understand the role of the mechanical interaction of the attachment proteins CD4 and E. coli pilus type 1. We have used atomic force microscopy to fully characterize the effect of mechanical forces in the structure, stability and chemistry of these proteins. We provide evidences of the role of their mechanical features in the initial stages of HIV-1 and UPEC infections. We have also developed novel procedures for searching small molecules that modify the mechanical properties of CD4, generating thus potential mechanoactive molecules that could impair the ability the organisms to attach and infect. Finally we have assembled two magnetic tweezers set up that allows us to visualize cells while they are under the effect of force exerted by magnetics beads. Overall, we have completed most of our objectives and several reports have been published with two more under preparation. In what follows we provide a brief summeary of the results per objective:
1. Understand the nanomechanics of CD4 and bacterial pilus type-1. We used AFM to apply mechanical forces from 10 to 1000 pN to investigate the mechanical stability, mechanical kinetics and mechanical hierarchies of viral receptor CD4, and bacterial pilus type-1.
Progress and results for this objective: We have extensively studied the mechanical properties of CD4 and bacterial pilus type-1. We have used single-molecule force spectroscopy to provide a complete nanomechanical description of domains D1 and D2 of CD4 and all the domains, FimA, FimF, FimG and FimH of the bacterial pilus from UPEC. Our work includes experiments of protein stretching to determine mechanical stability. We demonstrated that force may trigger conformational changes in CD4 domains that might be important during viral-receptor interaction. The CD4 results were published in Perez-Jimenez et al. ACS Nano, 2014, 8 (10), pp 10313–10320. In the case of pilus domains, we have discovered that their mechanical stability is amongst the higher ever reported in a protein. The results were published in Alonso-Caballero et al. Nature Comm, 2018, 9:2758.
2. Understand the mechanochemistry of CD4 and bacterial pilus type-1. Attachment proteins contain disulfide bonds that interact with enzymes such as thioredoxin and Dsb. This redox regulation is known to be important during viral infection in the case of CD4, and crucial for the strucrural integrity in bacterial pili. We have investigated the mechanochemistry of this redox regulation. Viral receptors also interact with ligands and antibodies. We have investigated whether this interaction has a mechanical component studying an antibody againts CD4 that have already shown efectiveness against HIV-1 infection. Finally, we have studied the hydrophobic interactions that mantain the integrity of fimbrial proteins from the pilus.
Progress and results for this objective: In the case of CD4 we have studied its interaction with thioredoxin enzymes and with the antibody Ibalizumab. Redox regulation of gp120 and CD4 seem to be important during HIV-1 infection. We have studied the interaction of thioreodixn, a key oxidoreductase enzyme, demonstrating that access of thioredoxin to CD4 disulfides for redox exchange requires mechanical exposure of the disulfide. This is a novel observation that demonstrates the importance of mechanical force as a main factor triggering conformational alterations in CD4 promote the oxidation/reduction of cryptic disulfide bonds. We also demonstrated that an antibody that is known to block HIV-1 entry makes CD4 more rigid by incresing the mechanical stability of the domains, probably restricting the conformational changes of CD4/gp120 interaction. In the case of bacterial pilus domains, we have discovered that disulfide bonds in Fim domains act not only as mechanical lockers but also increase the total mechanical stability of the protein by 30%. These disulfide bonds are oxidized by DsbA enzyme in the bacterial periplasm. We have studied the oxidative folding process assisted by DsbA and have discovered that this enzyme not only act as oxidoreductase but also as a molecular chaperon speeding up folding of the domains.
3. Discovery of molecules that alter the mechanics of CD4 and bacterial pilus type-1. We used a multidisciplinary approach combining experimental and computational methods to search and design small molecules and ligands that alter the nanomechanics of viral and bacterial anchoring proteins. We are using identification and molecular docking software to find druggable sites in these proteins and search for ligands that bind to them with the expectation that they will modify the nanomechanics of the attachment proteins. In addition, we screen virtual and experimental chemical libraries in seach of small molecules that alter the nanomechanics of these proteins.
Progress and results for this objective: We have set up a procedure for the identification of potential mechanoactive compounds for CD4 domains. We have used Schrödinger Suite (Glyde) to search virtual chemical libraries to identify small molecules that bind CD4 in mechanically relevant regions. The process requires, AFM data, molecular dynamics to identify potential target regions. The novelty of this procedure is that is not a blind search in the entire molecule, but rather a localize search in specific regions that are important for the overall mechanical rigidity of CD4. So far, we have identified 3 potential molecules that bind CD4 domains whit high energies. These molecules were purchased and tested in real AFM experiments providing clear evidence that they make CD4 domains more rigid. We call this molecules SUREMEL (Surface Receptor Mechanical Lockers). We are currently testing this molecules against HIV-1 viruses.
Bacteria and viruses infect organisms by using proteins that attach to molecules in the surface of the host. HIV/AIDS affects over 30 million people worldwide. HIV-1 uses its envelope glycoprotein gp120 to attach to CD4 in the surface of T cell. Similarly, the bacterium E. coli uses an array of proteins called pilus for establishing mechanical anchoring to tissues. These proteins withstand mechanical forces that go from few to hundreds of picoNewtons. The effect of these forces in the structure and chemistry of the proteins is not understood but it may have implications in the infection process. In this project, we have investigated the role of mechanical forces in the structure and chemistry of the microbial attachment proteins as well as the infection process. We have used an array of techniques to study the nanomechanics of viral and bacterial infections progressively from single molecules to cells. We have established new knowledge of the molecular aspects that drive the mechanical interaction of microbes with their targets. First, we have used atomic force spectroscopy to explore the effect of mechanical forces in microbial attachment proteins, human CD4 and E. coli pilus. This technique allows monitoring chemical reactions under force such as the reduction of disulfide bonds or the binding of peptides, small molecules and antibodies, processes which are known to be implicated in microbial infections and that may have a mechanical origin. Second, we have used bioinformatics to search for molecules that alter the mechanical properties of these anchoring proteins and that can potentially be used to prevent infections. Viral and bacterial infections have been widely studied but there is not much information in the context of mechanical interactions. We believe that our project is providing a new understanding about infectious diseases.
Under this grant we have made substantial progress to understand the role of the mechanical interaction of the attachment proteins CD4 and E. coli pilus type 1. We have used atomic force microscopy to fully characterize the effect of mechanical forces in the structure, stability and chemistry of these proteins. We provide evidences of the role of their mechanical features in the initial stages of HIV-1 and UPEC infections. We have also developed novel procedures for searching small molecules that modify the mechanical properties of CD4, generating thus potential mechanoactive molecules that could impair the ability the organisms to attach and infect. Finally we have assembled two magnetic tweezers set up that allows us to visualize cells while they are under the effect of force exerted by magnetics beads. Overall, we have completed most of our objectives and several reports have been published with two more under preparation. In what follows we provide a brief summeary of the results per objective:
1. Understand the nanomechanics of CD4 and bacterial pilus type-1. We used AFM to apply mechanical forces from 10 to 1000 pN to investigate the mechanical stability, mechanical kinetics and mechanical hierarchies of viral receptor CD4, and bacterial pilus type-1.
Progress and results for this objective: We have extensively studied the mechanical properties of CD4 and bacterial pilus type-1. We have used single-molecule force spectroscopy to provide a complete nanomechanical description of domains D1 and D2 of CD4 and all the domains, FimA, FimF, FimG and FimH of the bacterial pilus from UPEC. Our work includes experiments of protein stretching to determine mechanical stability. We demonstrated that force may trigger conformational changes in CD4 domains that might be important during viral-receptor interaction. The CD4 results were published in Perez-Jimenez et al. ACS Nano, 2014, 8 (10), pp 10313–10320. In the case of pilus domains, we have discovered that their mechanical stability is amongst the higher ever reported in a protein. The results were published in Alonso-Caballero et al. Nature Comm, 2018, 9:2758.
2. Understand the mechanochemistry of CD4 and bacterial pilus type-1. Attachment proteins contain disulfide bonds that interact with enzymes such as thioredoxin and Dsb. This redox regulation is known to be important during viral infection in the case of CD4, and crucial for the strucrural integrity in bacterial pili. We have investigated the mechanochemistry of this redox regulation. Viral receptors also interact with ligands and antibodies. We have investigated whether this interaction has a mechanical component studying an antibody againts CD4 that have already shown efectiveness against HIV-1 infection. Finally, we have studied the hydrophobic interactions that mantain the integrity of fimbrial proteins from the pilus.
Progress and results for this objective: In the case of CD4 we have studied its interaction with thioredoxin enzymes and with the antibody Ibalizumab. Redox regulation of gp120 and CD4 seem to be important during HIV-1 infection. We have studied the interaction of thioreodixn, a key oxidoreductase enzyme, demonstrating that access of thioredoxin to CD4 disulfides for redox exchange requires mechanical exposure of the disulfide. This is a novel observation that demonstrates the importance of mechanical force as a main factor triggering conformational alterations in CD4 promote the oxidation/reduction of cryptic disulfide bonds. We also demonstrated that an antibody that is known to block HIV-1 entry makes CD4 more rigid by incresing the mechanical stability of the domains, probably restricting the conformational changes of CD4/gp120 interaction. In the case of bacterial pilus domains, we have discovered that disulfide bonds in Fim domains act not only as mechanical lockers but also increase the total mechanical stability of the protein by 30%. These disulfide bonds are oxidized by DsbA enzyme in the bacterial periplasm. We have studied the oxidative folding process assisted by DsbA and have discovered that this enzyme not only act as oxidoreductase but also as a molecular chaperon speeding up folding of the domains.
3. Discovery of molecules that alter the mechanics of CD4 and bacterial pilus type-1. We used a multidisciplinary approach combining experimental and computational methods to search and design small molecules and ligands that alter the nanomechanics of viral and bacterial anchoring proteins. We are using identification and molecular docking software to find druggable sites in these proteins and search for ligands that bind to them with the expectation that they will modify the nanomechanics of the attachment proteins. In addition, we screen virtual and experimental chemical libraries in seach of small molecules that alter the nanomechanics of these proteins.
Progress and results for this objective: We have set up a procedure for the identification of potential mechanoactive compounds for CD4 domains. We have used Schrödinger Suite (Glyde) to search virtual chemical libraries to identify small molecules that bind CD4 in mechanically relevant regions. The process requires, AFM data, molecular dynamics to identify potential target regions. The novelty of this procedure is that is not a blind search in the entire molecule, but rather a localize search in specific regions that are important for the overall mechanical rigidity of CD4. So far, we have identified 3 potential molecules that bind CD4 domains whit high energies. These molecules were purchased and tested in real AFM experiments providing clear evidence that they make CD4 domains more rigid. We call this molecules SUREMEL (Surface Receptor Mechanical Lockers). We are currently testing this molecules against HIV-1 viruses.