scholarly journals Structural determinants for protein unfolding and translocation by the Hsp104 protein disaggregase

2017 ◽  
Vol 37 (6) ◽  
Author(s):  
Jungsoon Lee ◽  
Nuri Sung ◽  
Lythou Yeo ◽  
Changsoo Chang ◽  
Sukyeong Lee ◽  
...  
Keyword(s):  
Protein Unfolding ◽  
Substrate Binding ◽  
Binding Domain ◽  
Atp Binding ◽  
Structural Determinants ◽  
Functional Studies ◽  
Loop 2 ◽  
Loop Conformation ◽  
Do So

The ring-forming Hsp104 ATPase cooperates with Hsp70 and Hsp40 molecular chaperones to rescue stress-damaged proteins from both amorphous and amyloid-forming aggregates. The ability to do so relies upon pore loops present in the first ATP-binding domain (AAA-1; loop-1 and loop-2 ) and in the second ATP-binding domain (AAA-2; loop-3) of Hsp104, which face the protein translocating channel and couple ATP-driven changes in pore loop conformation to substrate translocation. A hallmark of loop-1 and loop-3 is an invariable and mutational sensitive aromatic amino acid (Tyr257 and Tyr662) involved in substrate binding. However, the role of conserved aliphatic residues (Lys256, Lys258, and Val663) flanking the pore loop tyrosines, and the function of loop-2 in protein disaggregation has not been investigated. Here we present the crystal structure of an N-terminal fragment of Saccharomyces cerevisiae Hsp104 exhibiting molecular interactions involving both AAA-1 pore loops, which resemble contacts with bound substrate. Corroborated by biochemical experiments and functional studies in yeast, we show that aliphatic residues flanking Tyr257 and Tyr662 are equally important for substrate interaction, and abolish Hsp104 function when mutated to glycine. Unexpectedly, we find that loop-2 is sensitive to aspartate substitutions that impair Hsp104 function and abolish protein disaggregation when loop-2 is replaced by four aspartate residues. Our observations suggest that Hsp104 pore loops have non-overlapping functions in protein disaggregation and together coordinate substrate binding, unfolding, and translocation through the Hsp104 hexamer.

scholarly journals The Role of the Conserved Glycines of ATP-binding Cassette Signature Motifs of MRP1 in the Communication between the Substrate-binding Site and the Catalytic Centers

2004 ◽  
Vol 279 (40) ◽  
pp. 41670-41678 ◽  
Author(s):  
Zsófia Szentpétery ◽  
András Kern ◽  
Károly Liliom ◽  
Balázs Sarkadi ◽  
András Váradi ◽  
...  
Keyword(s):  
Binding Site ◽  
Substrate Binding ◽  
Atp Binding ◽  
Substrate Binding Site ◽  
Atp Binding Cassette

Substrate Binding Causes Movement in the ATP Binding Domain ofEscherichia coliAdenylate Kinase†

Biochemistry ◽  
1996 ◽  
Vol 35 (19) ◽  
pp. 6100-6106 ◽  
Author(s):  
Tim Bilderback ◽  
Tim Fulmer ◽  
William W. Mantulin ◽  
Michael Glaser
Keyword(s):  
Substrate Binding ◽  
Binding Domain ◽  
Atp Binding

A Three-Dimensional Model for the Substrate Binding Domain of the Multidrug ATP Binding Cassette Transporter LmrA

2004 ◽  
Vol 66 (5) ◽  
pp. 1169-1179 ◽  
Author(s):  
Gerhard F. Ecker ◽  
Karin Pleban ◽  
Stephan Kopp ◽  
Edina Csaszar ◽  
Gerrit J. Poelarends ◽  
...  
Keyword(s):  
Substrate Binding ◽  
Three Dimensional ◽  
Binding Domain ◽  
Atp Binding ◽  
Dimensional Model ◽  
Three Dimensional Model ◽  
Atp Binding Cassette Transporter ◽  
Substrate Binding Domain ◽  
Atp Binding Cassette

scholarly journals Insight into the mechanism of H+-coupled nucleobase transport

2021 ◽  
Author(s):  
Jun Weng ◽  
Xiaoming Zhou ◽  
Pattama Wiriyasermkul ◽  
Zhenning Ren ◽  
Xiuwen Yan ◽  
...  
Keyword(s):  
Ascorbic Acid ◽  
Binding Site ◽  
Substrate Binding ◽  
Critical Role ◽  
Binding Domain ◽  
Purine Base ◽  
Substrate Binding Site ◽  
Functional Studies ◽  
Dependent Transport ◽  
Substrate Binding Domain

Members of the nucleobase/ascorbic acid transporter (NAT) gene family are found in all kingdoms of life. In mammals, the concentrative uptake of ascorbic acid (vitamin C) by members of the NAT family is driven by the Na+ gradient, while the uptake of nucleobases in bacteria is powered by the H+ gradient. Here we report the structure and function PurTCp, a NAT family member from Colwellia psychrerythraea. The structure of PurTCp was determined to 2.80 Å resolution by X-ray crystallography. PurTCp forms a homodimer and each protomer has 14 transmembrane segments folded into a substrate-binding domain (core domain) and an interface domain (gate domain) A purine base is present in the structure and defines the location of the substrate binding site. Functional studies reveal that PurTCp transports purines but not pyrimidines, and that purine binding and transport is dependent on the pH. Mutation of a conserved aspartate residue close to the substrate binding site reveals the critical role of this residue in H+-dependent transport of purines. Comparison of the PurTCp structure with transporters of the same structural fold suggests that rigid-body motions of the substrate-binding domain are central for substrate translocation across the membrane.

New crystal structures of HSC-70 ATP binding domain confirm the role of individual binding pockets and suggest a new method of inhibition

Biochimie ◽  
2015 ◽  
Vol 108 ◽  
pp. 186-192 ◽  
Author(s):  
Ziming Zhang ◽  
Jason Cellitti ◽  
Peter Teriete ◽  
Maurizio Pellecchia ◽  
Boguslaw Stec
Keyword(s):  
Crystal Structures ◽  
Binding Domain ◽  
New Method ◽  
Atp Binding ◽  
Hsc 70 ◽  
Binding Pockets

Recombinant preparation and functional studies of EspI ATP binding domain from Mycobacterium tuberculosis

2016 ◽  
Vol 123 ◽  
pp. 51-59 ◽  
Author(s):  
Hanyu Chen ◽  
Huilin Wang ◽  
Tao Sun ◽  
Shuangliang Tian ◽  
Donghai Lin ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Binding Domain ◽  
Atp Binding ◽  
Functional Studies

Abstract 1055: The 70 kDa Heat Shock Cognate Protein Plays A Novel Role In Myocardial Chemokine Expression And Cardiac Dysfunction Following Ischemia And Reperfusion Injury

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Ning Zou ◽  
Lihua Ao ◽  
Xiaoping Yang ◽  
Xin Su ◽  
David A Fullerton ◽  
...  
Keyword(s):  
Heat Shock ◽  
Inflammatory Response ◽  
Cardiac Dysfunction ◽  
Substrate Binding ◽  
Binding Domain ◽  
Chemokine Expression ◽  
Heat Shock Cognate Protein ◽  
Substrate Binding Domain ◽  
Cognate Protein

Myocardial ischemia and reperfusion (I/R) causes the release of cellular proteins including heat shock proteins (HSPs). The inducible HSP70 induces cytokine production in monocytes and dendritic cells through Toll-like receptor 4 (TLR4) signaling. In evaluation of the role of HSPs in myocardial inflammatory response in a mouse heart global I/R model, we found that the 70 kDa heat shock cognate protein (HSC70), but not HSP70, was released into the extracellular space and the coronary effluent during I/R. These observations prompted us to hypothesize that HSC70 plays a novel role in postischemic myocardial inflammatory response and cardiac dysfunction. Methods: We subjected mouse hearts to global I/R (20 min/60 min) or perfusion using the Langendorff method. We examined: the effect of HSC70 antibody on myocardial chemokine expression and cardiac functional recovery following I/R, the effect of recombinant HSC70 on myocardial chemokine expression and cardiac function and the role of TLR4 and the HSC70 substrate-binding domain in the effect of HSC70 on the heart. Results: In comparison with non-immune IgG, anti-HSC70 reduced myocardial expression of KC and MCP-1 mRNAs and proteins following I/R. Moreover, treatment with anti-HSC70 improved postischemic cardiac functional recovery (66±5.4% of baseline vs. 28±5.1% of baseline in hearts treated with non-immune IgG, p<0.01). Recombinant HSC70 induced myocardial expression of KC and MCP-1 mRNAs and proteins and caused cardiac dysfunction (72±2.6% of baseline vs. 98±3.9% of baseline in perfusion controls, p<0.001) in hearts with competent TLR4 (C3H/HeN). Interestingly, these effects of HSC70 were abrogated in hearts with defective TLR4 (C3H/HeJ). The potency of HSC70 was completely lost in the absence of its substrate-binding domain. Conclusions: Taken together, our studies demonstrate, for the first time, that HSC70 plays an important role in the induction of myocardial chemokines and cardiac dysfunction during I/R. The effect of HSC70 is dependent on TLR4 and requires the presence of the substrate-binding domain. The results suggest that the release of HSC70 into the extracellular space elicits an inflammatory response and causes mechanic dysfunction in the heart.

scholarly journals Role of the ATP-Binding Domain of the Human Papillomavirus Type 11 E1 Helicase in E2-Dependent Binding to the Origin

1999 ◽  
Vol 73 (7) ◽  
pp. 5282-5293 ◽  
Author(s):  
Steve Titolo ◽  
Alex Pelletier ◽  
Frédéric Sauvé ◽  
Karine Brault ◽  
Elizabeth Wardrop ◽  
...  
Keyword(s):  
Structural Integrity ◽  
Binding Domain ◽  
Human Papillomaviruses ◽  
Atp Binding ◽  
Bovine Papillomavirus ◽  
Structural Constraints ◽  
Transactivation Domain ◽  
Phosphate Binding

ABSTRACT Replication of the genome of human papillomaviruses (HPV) is initiated by the recruitment of the viral E1 helicase to the origin of DNA replication by the viral E2 protein, which binds specifically to the origin. We determined, for HPV type 11 (HPV-11), that the C-terminal 296 amino acids of E1 are sufficient for interaction with the transactivation domain of E2 in the yeast two-hybrid system and in vitro. This region of E1 encompasses the ATP-binding domain. Here we have examined the role of this ATP-binding domain, and of ATP, on E2-dependent binding of E1 to the origin. Several amino acid substitutions in the phosphate-binding loop (P loop), which is implicated in binding the triphosphate moiety of ATP, abolished E2 binding, indicating that the structural integrity of this domain is essential for the interaction. The structural constraints imposed on the E1 P loop may differ between HPV-11 and bovine papillomavirus type 1 (BPV-1), since the P479S substitution that inactivates BPV-1 E1 is tolerated in the HPV-11 enzyme. Other substitutions in the E1 P loop, or in two other conserved motifs of the ATP-binding domain, were tolerated, indicating that ATP binding is not essential for interaction with E2. Nevertheless, ATP-Mg stimulated the E2-dependent binding of E1 to the origin in vitro. This stimulation was maximal at the physiological temperature (37°C) and did not require ATP hydrolysis. In contrast, ATP-Mg did not stimulate the E2-dependent binding to the origin of an E1 protein containing only the C-terminal domain (353 to 649) or that of mutant E1 proteins with alterations in the DNA-binding domain. These results are discussed in light of a model in which the E1 ATP-binding domain is required for formation of the E2-binding surface and can, upon the binding of ATP, facilitate and/or stabilize the interaction of E1 with the origin.

scholarly journals Localization of the IGF binding domain and evaluation of the role of cysteine residues in IGF binding in IGF binding protein-4

2001 ◽  
Vol 169 (1) ◽  
pp. 135-143 ◽  
Author(s):  
D Byun ◽  
S Mohan ◽  
DJ Baylink ◽  
X Qin
Keyword(s):  
Binding Protein ◽  
Binding Activity ◽  
Binding Domain ◽  
Single Mutation ◽  
Structural Determinants ◽  
Fold Reduction ◽  
Igf I ◽  
Igf Binding ◽  
Igf Binding Protein

Our previous findings suggest that binding of IGF binding protein-4 (IGFBP-4) to IGFs is essential for the inhibitory effect of IGFBP-4 on the activity of IGFs, both in vitro and in vivo. Therefore, understanding the structural determinants of IGF binding in IGFBP-4 is important to the general understanding of the biology of the IGF system. This study sought to further localize the IGF binding domain and to evaluate the role of Cys residues in IGF binding. Our data revealed that full-length IGFBP-4 peptides lacking the residues Leu(72)-Ser(91) or Leu(72)-His(74) or Gly(75)-Ser(91) failed to bind to IGF-I or IGF-II, whereas deletion of the residue Leu(72) or residues Met(80)-Ser(91) led to a 2- to 3-fold reduction in IGF-I and IGF-II binding activity. The IGF-I and IGF-II binding activities were dramatically reduced by the single mutation, Cys9/Arg (>25-fold), and to a lesser degree, by the single mutation, Cys12/Arg (the first N-terminal Cys residue was designated Cys1). The mutation Cys17/Ser or Cys18/Tyr or Cys20/Ser each resulted in a similar but moderate ( approximately 5-fold) reduction in IGF-II binding activity. The IGF-I binding activity was also dramatically reduced by the mutation Cys18/Tyr, and to a lesser extent, by the mutation Cys17/Ser or Cys20/Ser. These data suggest: 1) the IGF-I and IGF-II binding domain in IGFBP-4 involves a hydrophobic motif (Leu(72)-Met(80)) located in the distal part of the conserved N-terminal region, and 2) the N-terminal Cys residues (Cys9 and Cys12) are more critical than the C-terminal Cys residues (Cys17 and Cys20) in affecting the IGF-I and IGF-II binding. Based on these data, we speculate that the structural determinants of IGF-I and IGF-II binding in IGFBP-4 are very similar, if not identical.

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