Cryo-EM of kinesin-binding protein: challenges and opportunities from protein-surface interactions

TY - JOUR

T1 - Cryo-EM of kinesin-binding protein

T2 - challenges and opportunities from protein-surface interactions

AU - Atherton, Joseph

AU - Moores, Carolyn A

N1 - open access.

PY - 2021/4/1

Y1 - 2021/4/1

N2 - Kinesin-binding protein (KBP) is an important selective inhibitor of specific kinesin family members and its genetic disruption causes Goldberg-Shprintzen syndrome. Cryo-electron microscopy (cryo-EM) has recently been used to reveal the structure of KBP alone (72 kDa) and in complex with the motor domain of the mitotic kinesin-12 KIF15 (110 kDa). KBP is an α-solenoid, tetratricopeptide-repeat protein that interacts with the microtubule-binding region of the kinesin motor domain and blocks microtubule attachment. Numerous challenges arose relating to the behavior of KBP and KBP-kinesin complexes during cryo-EM sample preparation. These included the partial denaturation of KBP by air-water interfaces, protein aggregation resulting from carbon interaction and preferential orientation. Sample preparation with a graphene oxide substrate enabled the eventual structure determination. Here, experiences with preparing these samples are detailed, bringing attention to some of the challenges and opportunities that are likely to arise from protein-surface interactions.

AB - Kinesin-binding protein (KBP) is an important selective inhibitor of specific kinesin family members and its genetic disruption causes Goldberg-Shprintzen syndrome. Cryo-electron microscopy (cryo-EM) has recently been used to reveal the structure of KBP alone (72 kDa) and in complex with the motor domain of the mitotic kinesin-12 KIF15 (110 kDa). KBP is an α-solenoid, tetratricopeptide-repeat protein that interacts with the microtubule-binding region of the kinesin motor domain and blocks microtubule attachment. Numerous challenges arose relating to the behavior of KBP and KBP-kinesin complexes during cryo-EM sample preparation. These included the partial denaturation of KBP by air-water interfaces, protein aggregation resulting from carbon interaction and preferential orientation. Sample preparation with a graphene oxide substrate enabled the eventual structure determination. Here, experiences with preparing these samples are detailed, bringing attention to some of the challenges and opportunities that are likely to arise from protein-surface interactions.

KW - Carrier Proteins/metabolism

KW - Cryoelectron Microscopy/methods

KW - Humans

KW - Kinesins/metabolism

KW - Models, Molecular

KW - Protein Binding

KW - Protein Domains

U2 - 10.1107/S2059798321001935

DO - 10.1107/S2059798321001935

M3 - Article

C2 - 33825702

SN - 2059-7983

VL - 77

SP - 411

EP - 423

JO - Acta Crystallographica Section D Structural Biology

JF - Acta Crystallographica Section D Structural Biology

IS - Pt 4

ER -