Functional analysis of proposed substrate-binding residues of Hsp104

The cellular machine Cdc48 functions in multiple biological pathways by segregating its protein substrates from a variety of stable environments such as organelles or multi-subunit complexes. Despite extensive studies, the mechanism of Cdc48 has remained obscure, and its reported structures are inconsistent with models of substrate translocation proposed for other AAA+ ATPases (adenosine triphosphatases). Here, we report a 3.7-angstrom–resolution structure of Cdc48 in complex with an adaptor protein and a native substrate. Cdc48 engages substrate by adopting a helical configuration of substrate-binding residues that extends through the central pore of both of the ATPase rings. These findings indicate a unified hand-over-hand mechanism of protein translocation by Cdc48 and other AAA+ ATPases.

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Crystal Structures of Yeast β-Alanine Synthase Complexes Reveal the Mode of Substrate Binding and Large Scale Domain Closure Movements

Journal of Biological Chemistry ◽

10.1074/jbc.m705517200 ◽

2007 ◽

Vol 282 (49) ◽

pp. 36037-36047 ◽

Cited By ~ 21

Author(s):

Stina Lundgren ◽

Birgit Andersen ◽

Jure Piškur ◽

Doreen Dobritzsch

Keyword(s):

Crystal Structures ◽

Large Scale ◽

Catalytic Domain ◽

Substrate Binding ◽

Salt Bridge ◽

Site Directed Mutagenesis ◽

Wild Type ◽

Dimerization Domain ◽

Binding Residues ◽

Present Site

β-Alanine synthase is the final enzyme of the reductive pyrimidine catabolic pathway, which is responsible for the breakdown of uracil and thymine in higher organisms. The fold of the homodimeric enzyme from the yeast Saccharomyces kluyveri identifies it as a member of the AcyI/M20 family of metallopeptidases. Its subunit consists of a catalytic domain harboring a di-zinc center and a smaller dimerization domain. The present site-directed mutagenesis studies identify Glu159 and Arg322 as crucial for catalysis and His262 and His397 as functionally important but not essential. We determined the crystal structures of wild-type β-alanine synthase in complex with the reaction product β-alanine, and of the mutant E159A with the substrate N-carbamyl-β-alanine, revealing the closed state of a dimeric AcyI/M20 metallopeptidase-like enzyme. Subunit closure is achieved by a ∼30° rigid body domain rotation, which completes the active site by integration of substrate binding residues that belong to the dimerization domain of the same or the partner subunit. Substrate binding is achieved via a salt bridge, a number of hydrogen bonds, and coordination to one of the zinc ions of the di-metal center.

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Structural and functional analysis of Oceanobacillus iheyensis macrodomain reveals a network of waters involved in substrate binding and catalysis

Open Biology ◽

10.1098/rsob.160327 ◽

2017 ◽

Vol 7 (4) ◽

pp. 160327 ◽

Cited By ~ 6

Author(s):

Rubén Zapata-Pérez ◽

Fernando Gil-Ortiz ◽

Ana Belén Martínez-Moñino ◽

Antonio Ginés García-Saura ◽

Jordi Juanhuix ◽

...

Keyword(s):

Functional Analysis ◽

Enzyme Activity ◽

Substrate Binding ◽

Therapeutic Agents ◽

Water Molecules ◽

Substrate Conformation ◽

Coordinated Water ◽

Alkaliphilic Bacterium ◽

Hydrolysis Of

Macrodomains are ubiquitous conserved domains that bind or transform ADP-ribose (ADPr) metabolites. In humans, they are involved in transcription, X-chromosome inactivation, neurodegeneration and modulating PARP1 signalling, making them potential targets for therapeutic agents. Unfortunately, some aspects related to the substrate binding and catalysis of MacroD-like macrodomains still remain unclear, since mutation of the proposed catalytic aspartate does not completely abolish enzyme activity. Here, we present a functional and structural characterization of a macrodomain from the extremely halotolerant and alkaliphilic bacterium Oceanobacillus iheyensis (OiMacroD), related to hMacroD1/hMacroD2, shedding light on substrate binding and catalysis. The crystal structures of D40A, N30A and G37V mutants, and those with MES, ADPr and ADP bound, allowed us to identify five fixed water molecules that play a significant role in substrate binding. Closure of the β6–α4 loop is revealed as essential not only for pyrophosphate recognition, but also for distal ribose orientation. In addition, a novel structural role for residue D40 is identified. Furthermore, it is revealed that OiMacroD not only catalyses the hydrolysis of O -acetyl-ADP-ribose but also reverses protein mono-ADP-ribosylation. Finally, mutant G37V supports the participation of a substrate-coordinated water molecule in catalysis that helps to select the proper substrate conformation.

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