Substrate Binding StabilizesS-Adenosylhomocysteine Hydrolase in a Closed Conformation†

Biochemistry ◽  
2000 ◽  
Vol 39 (32) ◽  
pp. 9811-9818 ◽  
Author(s):  
Dan Yin ◽  
Xiaoda Yang ◽  
Yongbo Hu ◽  
Krysztof Kuczera ◽  
Richard L. Schowen ◽  
...  
Keyword(s):  
Substrate Binding ◽  
Adenosylhomocysteine Hydrolase ◽  
Closed Conformation

scholarly journals Functional role of residues involved in substrate binding of human 4-hydroxyphenylpyruvate dioxygenase

2021 ◽  
Author(s):  
Chih-Wei Huang ◽  
Chi-Ching Hwang ◽  
Yung-Lung Chang ◽  
Jen-Tzu Liu ◽  
Sheng-Peng Wu ◽  
...  
Keyword(s):  
Binding Affinity ◽  
Functional Role ◽  
Substrate Binding ◽  
Critical Role ◽  
Intermediate Formation ◽  
Hydroxyl Group ◽  
Double Mutation ◽  
Closed Conformation ◽  
Bond Network

4-Hydroxylphenylpyruvate dioxygenase (HPPD) catalyzes the conversion of 4-hydroxylphenylpyruvate (HPP) to homogentisate, the important step for tyrosine catabolism. Comparison of the structure of human HPPD with the substrate-bound structure of A. thaliana HPPD revealed notably different orientations of the C-terminal helix. This helix performed as a closed conformation in human enzyme. Simulation revealed a different substrate-binding mode in which the carboxyl group of HPP interacted by a H-bond network formed by Gln334, Glu349 (the metal-binding ligand), and Asn363 (in the C-terminal helix). The 4-hydroxyl group of HPP interacted with Gln251 and Gln265. The relative activity and substrate-binding affinity were preserved for the Q334A mutant, implying the alternative role of Asn363 for HPP binding and catalysis. The reduction in kcat/Km of the Asn363 mutants confirmed the critical role in catalysis. Compared to the N363A mutant, the dramatic reduction in the Kd and thermal stability of the N363D mutant implies the side-chain effect in the hinge region rotation of the C-terminal helix. The activity and binding affinity were not recovered by double mutation; however, the 4-hydroxyphenylacetate intermediate formation by the uncoupled reaction of Q334N/N363Q and Q334A/N363D mutants indicated the importance of the H-bond network in the electrophilic reaction. These results highlight the functional role of the H-bond network in a closed conformation of the C-terminal helix to stabilize the bound substrate. The extremely low activity and reduction in Q251E’s Kd suggest that interaction coupled with the H-bond network is crucial to locate the substrate for nucleophilic reaction.

scholarly journals Not Cleaving the His-tag of Thal Results in More Tightly Packed and Better-Diffracting Crystals

Crystals ◽  
2020 ◽  
Vol 10 (12) ◽  
pp. 1135
Author(s):  
Ann-Christin Moritzer ◽  
Tina Prior ◽  
Hartmut H. Niemann
Keyword(s):  
Active Site ◽  
Flavin Adenine Dinucleotide ◽  
Substrate Binding ◽  
Environmentally Friendly ◽  
Space Group ◽  
Cofactor Binding ◽  
Closed Conformation ◽  
His Tag ◽  
Halide Salts ◽  
Binding Loop

Flavin-dependent halogenases chlorinate or brominate their substrates in an environmentally friendly manner, only requiring the cofactor reduced flavin adenine dinucleotide (FADH2), oxygen, and halide salts. The tryptophan 6-halogenase Thal exhibits two flexible loops, which become ordered (substrate-binding loop) or adopt a closed conformation (FAD loop) upon substrate or cofactor binding. Here, we describe the structure of NHis-Thal-RebH5 containing an N-terminal His-tag from pET28a, which crystallized in a different space group (P21) and, surprisingly, diffracted to a higher resolution of 1.63 Å than previously deposited Thal structures (P64; ~2.2 Å) with cleaved His-tag. Interestingly, the binding of glycine in the active site can induce an ordered conformation of the substrate-binding loop.

Computational Characterization of Substrate Binding and Catalysis inS-Adenosylhomocysteine Hydrolase†

Biochemistry ◽  
2001 ◽  
Vol 40 (50) ◽  
pp. 15143-15152 ◽  
Author(s):  
Yongbo Hu ◽  
Xiaoda Yang ◽  
Daniel H. Yin ◽  
Janaki Mahadevan ◽  
Krzysztof Kuczera ◽  
...  
Keyword(s):  
Substrate Binding ◽  
Adenosylhomocysteine Hydrolase

scholarly journals Conformational and dynamic plasticity in substrate-binding proteins underlies selective transport in ABC importers

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Marijn de Boer ◽  
Giorgos Gouridis ◽  
Ruslan Vietrov ◽  
Stephanie L Begg ◽  
Gea K Schuurman-Wolters ◽  
...  
Keyword(s):  
Single Molecule ◽  
Binding Proteins ◽  
Substrate Binding ◽  
Conformational Dynamics ◽  
Complex Interplay ◽  
Selective Transport ◽  
Closed Conformation ◽  
Single Molecule Fret ◽  
The Impact ◽  
Dynamic Plasticity

Substrate-binding proteins (SBPs) are associated with ATP-binding cassette importers and switch from an open to a closed conformation upon substrate binding, providing specificity for transport. We investigated the effect of substrates on the conformational dynamics of six SBPs and the impact on transport. Using single-molecule FRET, we reveal an unrecognized diversity of plasticity in SBPs. We show that a unique closed SBP conformation does not exist for transported substrates. Instead, SBPs sample a range of conformations that activate transport. Certain non-transported ligands leave the structure largely unaltered or trigger a conformation distinct from that of transported substrates. Intriguingly, in some cases, similar SBP conformations are formed by both transported and non-transported ligands. In this case, the inability for transport arises from slow opening of the SBP or the selectivity provided by the translocator. Our results reveal the complex interplay between ligand-SBP interactions, SBP conformational dynamics and substrate transport.

scholarly journals Structural basis for the mechanisms of human presequence protease conformational switch and substrate recognition

2020 ◽  
Author(s):  
Wenguang Liang ◽  
Juwina wijaya ◽  
Hui Wei ◽  
Alex Noble ◽  
Swansea Mo ◽  
...  
Keyword(s):  
Citrate Synthase ◽  
Substrate Binding ◽  
Substrate Recognition ◽  
Amyloid Β ◽  
Body Motion ◽  
Structural Basis ◽  
Conformational Switch ◽  
Closed Conformation ◽  
Air Water Interface ◽  
Open States

Abstract Presequence protease (PreP), a 117 kDa mitochondrial M16C metalloprotease vital for mitochondrial proteostasis, degrades presequence peptides cleaved off from nuclear-encoded proteins and other aggregation-prone peptides, such as amyloid β (Aβ). PreP structures have only been determined in a closed conformation; thus, the mechanisms of substrate binding and selectivity remain elusive. Here, we leverage advanced vitrification techniques to overcome the preferential denaturation of one of two ~55 kDa homologous domains of PreP caused by air-water interface adsorption, and thereby elucidate cryoEM structures of three apo-PreP open states along with Aβ- and citrate synthase presequence-bound PreP at 3.3 Å-4.6 Å resolution. Together with integrative biophysical and pharmacological approaches, these structures reveal the key stages of the PreP catalytic cycle and how the binding of substrates or PreP inhibitor drives the rigid body motion of the protein for substrate binding and catalysis. Together, our studies provide key mechanistic insights into M16C metalloproteases for future therapeutic innovations.

scholarly journals Structural characterization of theStreptococcus pneumoniaecarbohydrate substrate-binding protein SP0092

2017 ◽  
Vol 73 (1) ◽  
pp. 54-61 ◽  
Author(s):  
Simone Culurgioni ◽  
Minzhe Tang ◽  
Martin Austin Walsh
Keyword(s):  
Binding Protein ◽  
Substrate Binding ◽  
The Elderly ◽  
Binding Pocket ◽  
Respiratory Pathogen ◽  
Dimer Formation ◽  
Closed Conformation ◽  
Carbohydrate Ligand ◽  
Substrate Binding Protein

Streptococcus pneumoniaeis an opportunistic respiratory pathogen that remains a major cause of morbidity and mortality globally, with infants and the elderly at the highest risk.S. pneumoniaerelies entirely on carbohydrates as a source of carbon and dedicates a third of all uptake systems to carbohydrate import. The structure of the carbohydrate-free substrate-binding protein SP0092 at 1.61 Å resolution reveals it to belong to the newly proposed subclass G of substrate-binding proteins, with a ligand-binding pocket that is large enough to accommodate complex oligosaccharides. SP0092 is a dimer in solution and the crystal structure reveals a domain-swapped dimer with the monomer subunits in a closed conformation but in the absence of carbohydrate ligand. This closed conformation may be induced by dimer formation and could be used as a mechanism to regulate carbohydrate uptake.

scholarly journals Conformational and dynamical plasticity in substrate-binding proteins underlies selective transport in ABC importers

2019 ◽  
Author(s):  
Marijn de Boer ◽  
Giorgos Gouridis ◽  
Ruslan Vietrov ◽  
Stephanie L. Begg ◽  
Gea K. Schuurman-Wolters ◽  
...  
Keyword(s):  
Single Molecule ◽  
Binding Proteins ◽  
Substrate Binding ◽  
Conformational Dynamics ◽  
Complex Interplay ◽  
Substrate Transport ◽  
Selective Transport ◽  
Closed Conformation ◽  
Single Molecule Fret ◽  
The Impact

ABSTRACTSubstrate-binding proteins (SBPs) are associated with ATP-binding cassette importers and switch from an open-to a closed-conformation upon substrate binding providing specificity for transport. We investigated the effect of substrates on the conformational dynamics of six SBPs and the impact on transport. Using single-molecule FRET, we reveal an unrecognized diversity of plasticity in SBPs. We show that a unique closed SBP conformation does not exist for transported substrates. Instead, SBPs sample a range of conformations that activate transport. Certain non-transported ligands leave the structure largely unaltered or trigger a conformation distinct from that of transported substrates. Intriguingly, in some cases similar SBP conformations are formed by both transported and non-transported ligands. In this case, the inability for transport arises from slow opening of the SBP or the selectivity provided by the translocator. Our results reveal the complex interplay between ligand-SBP interactions, SBP conformational dynamics and substrate transport.

Fibronexus-Like Adhesion Sites are Present at the Invasive Zones of Human Epidermoid Carcinomas

1982 ◽  
Vol 40 ◽  
pp. 106-109
Author(s):  
Irwin I. Singer
Keyword(s):  
Cell Cycle ◽  
Serum Concentration ◽  
Substrate Binding ◽  
High Serum ◽  
Adhesion Sites ◽  
Substrate Adhesion ◽  
Focal Contacts ◽  
Adhesion Complex ◽  
Low Serum

Our previous results indicate that two types of fibronectin-cytoskeletal associations may be formed at the fibroblast surface: dorsal matrixbinding fibronexuses generated in high serum (5% FBS) cultures, and ventral substrate-adhering units formed in low serum (0.3% FBS) cultures. The substrate-adhering fibronexus consists of at least vinculin (VN) and actin in its cytoplasmic leg, and fibronectin (FN) as one of its major extracellular components. This substrate-adhesion complex is localized in focal contacts, the sites of closest substratum approach visualized with interference reflection microscopy, which appear to be the major points of cell-tosubstrate adhesion. In fibroblasts, the latter substrate-binding complex is characteristic of cultures that are arrested at the G1 phase of the cell cycle due to the low serum concentration in their medium. These arrested fibroblasts are very well spread, flattened, and immobile.

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