Spreading the "resolution revolution" across Latin American through Cryo-EM studies of a mollusk hemocyanin, with substantial biomedical impact

• Grant number: 19/13318-5
• Support Opportunities: Regular Research Grants
• Start date: August 01, 2020
• End date: January 31, 2023
• Field of knowledge: Biological Sciences - Biochemistry - Chemistry of Macromolecules
• Agreement: Comisión Nacional de Investigación Científica y Tecnológica (CONICYT)
• Principal Investigator: Andre Luis Berteli Ambrosio
• Grantee: Andre Luis Berteli Ambrosio
• Principal researcher abroad: Victoria Guixe
• Institution abroad: Universidad de Chile, Chile
• Host Institution: Instituto de Física de São Carlos (IFSC). Universidade de São Paulo (USP). São Carlos , SP, Brazil

Abstract

The outstanding progress in structural biology made possible by single particle cryo-electron microscopy (cryo-EM), originated the so-called "resolution revolution," mainly due to its ability to provide high-resolution structural information of large and dynamic protein complexes. Recently, Brazil has acquired a state-of-the-art Titan Krios microscope, which, when in operation at the Nanotechnology National Laboratory (LNNano), will establish this country as the leader in the field of cryo-EM in the Latin American. However, to optimize the use and occupation of such facility by the structural biology community, it is urgent to capacitate researchers in the laboratories across the region. Wide-spread access to this methodology, through collaborative efforts between research groups from Chile and Brazil, represent an invaluable opportunity to promote further regional scientific development employing a cutting-edge methodology. Here, we propose a synergistic approach to determine the cryo-EM structure of the hemocyanin from Concholepas concholepas (CCH), a marine organism endemic of the southern Pacific coasts. Hemocyanins are megadalton-sized oligomers that participate in oxygen transport in multiple invertebrate organisms. Currently, hemocyanins are used as natural non-toxic, and nonspecific immunostimulants with key biomedical and clinical applications; thus, understanding the precise molecular mechanisms of hemocyanins is essential for future targeted improvement. Currently, the most well-studied hemocyanin from a structural and immunological standpoint is KLH, from Megathura crenulate. Through a combination of cryo-EM and X-ray crystallography, the atomic details of the functional units that compose KLH have been revealed, displaying highly symmetric homodecameric rings featuring extensively glycosylated regions. However, studies of homologous hemocyanins from other marine organisms have proven that there is a significant degree of structural diversity among them, especially concerning glycosylation patterns, which might explain their unequal efficiency in generating appropriate adjuvant effects for immunological oriented therapies. Studies conducted by Chilean researchers, who are also associated with this proposal, recently showed that CCH is one of the most promising hemocyanins for biomedical applications. CCH has superior stability and solubility compared to homologs, and it is the most effective in inducing immunological responses, as observed in preclinical studies of cancer vaccines and immunocastration. Nevertheless, despite the unique biomedical potential of CCH, its structure and even its amino acid sequence remain unknown. Preliminary biochemical characterization of CCH suggested two distinct polypeptide chains forming heterodimeric rings, as opposed to the homodimeric rings of KLH. Each CCH subunit appears to have eight globular oxygen-binding domains, termed functional units (FU), which are differentially glycosylated. In this context, we propose three major goals: (i) to determine the CCH gene sequence through RACE sequencing, (ii) to determine the structure of at least one of its functional units by X-ray crystallography, and (iii) to determine the overall architecture of CCH through single particle cryo-EM. We seek to combine the access to Concholepas concholepas specimens and the expert knowledge regarding the molecular biology and biochemistry of this organism by the Chilean researchers, and the knowledge in structure determination by means of X-ray crystallography and cryo-EM of the Brazilian counterpart. An additional advantage on the Brazilian side is the substantially easier geographical access to sophisticated multiuser instrumentation dedicated to structural biology, such as the Titan Krios (at LNNano/CNPEM) and the SIRIUS fourth generation synchrotron source (at LNLS/CNPEM). Preliminary results that strengthen the proposal are the availability of partial gene sequences for both CCH chains, cleavage... (AU)