Mechanism of activation and regulation of Deubiquitinase activity in MINDY1 and MINDY2

TY - JOUR

T1 - Mechanism of activation and regulation of Deubiquitinase activity in MINDY1 and MINDY2

AU - Rehman, Syed Arif Abdul

AU - Armstrong, Lee A.

AU - Lange, Sven M.

AU - Kristariyanto, Yosua Adi

AU - Gräwert, Tobias W.

AU - Knebel, Axel

AU - Svergun, Dmitri I.

AU - Kulathu, Yogesh

N1 - We thank members of the Kulathu lab, specifically Drs Kwasna, Matthews and McFarland for discussions and critical comments on the manuscript, Dr. Garib Murshudov, MRC LMB for advice. We thank MRC Reagents and Services team, especially Simone Weidlich for the help with cloning. Crystallographic data were collected at the European Synchrotron Radiation Facility (ESRF) beamlines ID23-1, ID23-2, ID-29, ID30-A1, ID30B and at Diamond Light Source (DLS) beamline I03. The SAXS measurements were made at P12 beamline, PETRA III, DESY, Hamburg. TG was supported by the iNEXT project (653706) funded by the Horizon 2020 program of the European Commission. This work was supported by the Medical Research Council UK (MC_UU_00018/3), EMBO Young Investigator Programme, ERC Starting grant (677623).

PY - 2021/10/21

Y1 - 2021/10/21

N2 - Of the eight distinct polyubiquitin (polyUb) linkages that can be assembled, the roles of K48-linked polyUb (K48-polyUb) are the most established, with K48-polyUb modified proteins being targeted for degradation. MINDY1 and MINDY2 are members of the MINDY family of deubiquitinases (DUBs) that have exquisite specificity for cleaving K48-polyUb, yet we have a poor understanding of their catalytic mechanism. Here, we analyze the crystal structures of MINDY1 and MINDY2 alone and in complex with monoUb, di-, and penta-K48-polyUb, identifying 5 distinct Ub binding sites in the catalytic domain that explain how these DUBs sense both Ub chain length and linkage type to cleave K48-polyUb chains. The activity of MINDY1/2 is inhibited by the Cys-loop, and we find that substrate interaction relieves autoinhibition to activate these DUBs. We also find that MINDY1/2 use a non-canonical catalytic triad composed of Cys-His-Thr. Our findings highlight multiple layers of regulation modulating DUB activity in MINDY1 and MINDY2.

AB - Of the eight distinct polyubiquitin (polyUb) linkages that can be assembled, the roles of K48-linked polyUb (K48-polyUb) are the most established, with K48-polyUb modified proteins being targeted for degradation. MINDY1 and MINDY2 are members of the MINDY family of deubiquitinases (DUBs) that have exquisite specificity for cleaving K48-polyUb, yet we have a poor understanding of their catalytic mechanism. Here, we analyze the crystal structures of MINDY1 and MINDY2 alone and in complex with monoUb, di-, and penta-K48-polyUb, identifying 5 distinct Ub binding sites in the catalytic domain that explain how these DUBs sense both Ub chain length and linkage type to cleave K48-polyUb chains. The activity of MINDY1/2 is inhibited by the Cys-loop, and we find that substrate interaction relieves autoinhibition to activate these DUBs. We also find that MINDY1/2 use a non-canonical catalytic triad composed of Cys-His-Thr. Our findings highlight multiple layers of regulation modulating DUB activity in MINDY1 and MINDY2.

KW - ubiquitylation

KW - deubiquitinase

KW - crystal structure

KW - polyubiquitin

KW - protease

KW - enzyme mechanism

KW - protein degradation

KW - proteasome

KW - conformational change

KW - autoinhibition

UR - https://www.scopus.com/pages/publications/85119978562

U2 - 10.1016/j.molcel.2021.08.024

DO - 10.1016/j.molcel.2021.08.024

M3 - Article

C2 - 34529927

SN - 1097-2765

VL - 81

SP - 4176-4190.e6

JO - Molecular Cell

JF - Molecular Cell

IS - 20

ER -