Year: 2026
Type: preprint
Abstract: Cardiovascular diseases, including ischemic stroke, necessitate improved thrombolytic agents. A microbe-encoded plasminogen activator staphylokinase (SAK) is a promising alternative to the widely used tissue plasminogen activator (tPA) due to its high fibrin specificity and low production cost. To overcome potential immunogenicity hampering its use in clinical settings, the low-immunogenic variants SAK SY155 and SAK THR174 were previously engineered. However, the molecular basis underlying their reduced immunogenicity is not understood and requires detailed elucidation. Here, we determine molecular structures and compare flexibility between low-immunogenic and immunogenic SAK variants, using a combination of experimental and computational structural techniques. Our analyses show that all variants share the canonical SAK fold and retain similar plasminogen activation kinetics, despite the number of introduced substitutions. Crucially, the low-immunogenic variants exhibit distinct flexibility profiles, with SAK THR174 showing substantially increased flexibility in the H1 helix and B3 region. SAK SY155 exhibits an increased flexibility in the H1-B3 loop and propensity to homodimerize. These flexibility changes are found in the known immunogenic epitopes. Our multi-scale flexibility analysis provides the molecular explanation for the reduced immunogenicity, altered thermostability, and retained fibrinolytic function of the engineered variants. This information is critical for the design of next-generation thrombolytics.
Authors: Anthony P. Legrand, Linda Kasiarova, Naina Verma, Jan Mican, Joan Planas Iglesias, Pavel Kohout, Alan Strunga, Josef Kucera, Tomas Henek, Pavel Vanacek, Tim de Martines, Pavel Kaderavek, Lukas Zidek, Jiri Damborsky, Stanislav Mazurenko, David Bednar, Lenka Hernychova, Zbynek Prokop, Martin Marek
Institutions: Masaryk University, Masaryk Memorial Cancer Institute
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
Awards: RECETOX Research Infrastructure, G1761812275, G2020983025, CETOCOEN Excellence, G252385833, G2678520069, G2698300789, G291036901, Czech National Node of the European Clinical Research Infrastructures Network, G3735356012, G3850027771, Czech National Infrastructure for Biological Data, G434337789, G5652504370, Center for Artificial Intelligence and Quantum Computing in System Brain Research, Resolving the puzzle of resonance energy transfer in bioluminescence, G7534645696, G8227998441, Czech Infrastructure for Integrative Structural Biology, G941802975
OpenAlex ID: W7128220174