Journal: Nat Commun / Year: 2022 Title: Allosteric inhibition of HTRA1 activity by a conformational lock mechanism to treat age-related macular degeneration. Authors: Stefan Gerhardy / Mark Ultsch / Wanjian Tang / Evan Green / Jeffrey K Holden / Wei Li / Alberto Estevez / Chris Arthur / Irene Tom / Alexis Rohou / Daniel Kirchhofer / Abstract: The trimeric serine protease HTRA1 is a genetic risk factor associated with geographic atrophy (GA), a currently untreatable form of age-related macular degeneration. Here, we describe the allosteric ...The trimeric serine protease HTRA1 is a genetic risk factor associated with geographic atrophy (GA), a currently untreatable form of age-related macular degeneration. Here, we describe the allosteric inhibition mechanism of HTRA1 by a clinical Fab fragment, currently being evaluated for GA treatment. Using cryo-EM, X-ray crystallography and biochemical assays we identify the exposed LoopA of HTRA1 as the sole Fab epitope, which is approximately 30 Å away from the active site. The cryo-EM structure of the HTRA1:Fab complex in combination with molecular dynamics simulations revealed that Fab binding to LoopA locks HTRA1 in a non-competent conformational state, incapable of supporting catalysis. Moreover, grafting the HTRA1-LoopA epitope onto HTRA2 and HTRA3 transferred the allosteric inhibition mechanism. This suggests a conserved conformational lock mechanism across the HTRA family and a critical role of LoopA for catalysis, which was supported by the reduced activity of HTRA1-3 upon LoopA deletion or perturbation. This study reveals the long-range inhibition mechanism of the clinical Fab and identifies an essential function of the exposed LoopA for activity of HTRA family proteases.
Model: Quantifoil R0.6/1 / Material: GOLD / Mesh: 400 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.067 kPa Details: SAM: Grids were incubated in 4 mM monothiolalkane(C11)PEG6-OH (11-mercaptoundecyl) hexaethylenglycol (SPT-0011P6, SensoPath Technologies, Inc., Bozeman, MT) for 24h and rinsed in EtOH before sample application
Vitrification
Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: LEICA EM GP / Details: 3.5s blot time.
Details
HtrA1PD:Fab15H6.v4 complex
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Electron microscopy
Microscope
FEI TITAN KRIOS
Image recording
Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Number grids imaged: 1 / Number real images: 5345 / Average exposure time: 3.0 sec. / Average electron dose: 64.0 e/Å2
Electron beam
Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
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