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Title: Structural and functional characterization of the dominant negative P‐loop lysine mutation in the dynamin superfamily protein Vps1

Journal Article · · Protein Science
DOI: https://doi.org/10.1002/pro.3830 · OSTI ID:1596867
ORCiD logo [1]; ORCiD logo [1];  [2]; ORCiD logo [2]; ORCiD logo [1]
  1. Department of Cell Biology University of Pittsburgh School of Medicine Pittsburgh Pennsylvania
  2. Department of Molecular Medicine Cornell University Ithaca New York
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Abstract Dynamin‐superfamily proteins (DSPs) are large self‐assembling mechanochemical GTPases that harness GTP hydrolysis to drive membrane remodeling events needed for many cellular processes. Mutation to alanine of a fully conserved lysine within the P‐loop of the DSP GTPase domain results in abrogation of GTPase activity. This mutant has been widely used in the context of several DSPs as a dominant‐negative to impair DSP‐dependent processes. However, the precise deficit of the P‐loop K to A mutation remains an open question. Here, we use biophysical, biochemical and structural approaches to characterize this mutant in the context of the endosomal DSP Vps1. We show that the Vps1 P‐loop K to A mutant binds nucleotide with an affinity similar to wild type but exhibits defects in the organization of the GTPase active site that explain the lack of hydrolysis. In cells, Vps1 and Dnm1 bearing the P‐loop K to A mutation are defective in disassembly. These mutants become trapped in assemblies at the typical site of action of the DSP. This work provides mechanistic insight into the widely‐used DSP P‐loop K to A mutation and the basis of its dominant‐negative effects in the cell.

Sponsoring Organization:
USDOE
Grant/Contract Number:
W-31109-ENG-38; AC02-06CH11357
OSTI ID:
1596867
Journal Information:
Protein Science, Journal Name: Protein Science Journal Issue: 6 Vol. 29; ISSN 0961-8368
Publisher:
Wiley Blackwell (John Wiley & Sons)Copyright Statement
Country of Publication:
United Kingdom
Language:
English

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