scholarly journals A Glimpse into the Structural Properties of the Intermediate and Transition State in the Folding of Bromodomain 2 Domain 2 by Φ Value Analysis

2021 ◽  
Vol 22 (11) ◽  
pp. 5953
Author(s):  
Leonore Novak ◽  
Maria Petrosino ◽  
Daniele Santorelli ◽  
Roberta Chiaraluce ◽  
Valerio Consalvi ◽  
...  
Keyword(s):  
Transition State ◽  
Chromatin Remodeling ◽  
Mutational Analysis ◽  
Structural Information ◽  
Terminal Region ◽  
Folding Pathway ◽  
Value Analysis ◽  
Folding Nucleus ◽  
Late Transition ◽  
Partially Folded State

Bromodomains (BRDs) are small protein interaction modules of about 110 amino acids that selectively recognize acetylated lysine in histones and other proteins. These domains have been identified in a variety of multi-domain proteins involved in transcriptional regulation or chromatin remodeling in eukaryotic cells. BRD inhibition is considered an attractive therapeutic approach in epigenetic disorders, particularly in oncology. Here, we present a Φ value analysis to investigate the folding pathway of the second domain of BRD2 (BRD2(2)). Using an extensive mutational analysis based on 25 site-directed mutants, we provide structural information on both the intermediate and late transition state of BRD2(2). The data reveal that the C-terminal region represents part of the initial folding nucleus, while the N-terminal region of the domain consolidates its structure only later in the folding process. Furthermore, only a small number of native-like interactions have been identified, suggesting the presence of a non-compact, partially folded state with scarce native-like characteristics. Taken together, these results indicate that, in BRD2(2), a hierarchical mechanism of protein folding can be described with non-native interactions that play a significant role in folding.

Mutational investigation of protein folding transition states by Φ-value analysis and beyond: lessons from SH3 domain foldingThis paper is one of a selection of papers published in this special issue entitled “Canadian Society of Biochemistry, Molecular & Cellular Biology 52nd Annual Meeting — Protein Folding: Principles and Diseases” and has undergone the Journal's usual peer review process.

2010 ◽  
Vol 88 (2) ◽  
pp. 231-238 ◽  
Author(s):  
Arash Zarrine-Afsar ◽  
Sung Lun Lin ◽  
Philipp Neudecker
Keyword(s):  
Protein Folding ◽  
Transition State ◽  
Structural Information ◽  
Peer Review Process ◽  
Transition States ◽  
Nmr Relaxation ◽  
Relaxation Dispersion ◽  
Folding Kinetics ◽  
Value Analysis ◽  
Kinetic Effects

Understanding how proteins adopt their unique native structures requires a complete structural characterization of the rate-limiting transition state(s) along the folding pathway. By definition, transition states are not significantly populated and are only accessible via folding kinetics studies. In this respect, interpreting the kinetic effects of amino acid substitutions (especially to Ala) via Φ-value analysis is the most common method to probe the structure of these transient, yet important states. A critical review of the key assumptions required for rigorous interpretation of Φ values reveals that a multiple substitution strategy in which a position of interest is mutated to a variety of amino acids, and not exclusively to Ala, provides the best means to characterize folding transition states. This approach has proven useful in revealing non-native interactions and (or) conformations in folding transition states. Moreover, by simultaneously examining the folding kinetics of multiple substitutions made at a single surface-exposed position using the Brønsted analysis the backbone conformation in a folding transition state can be investigated. For folding equilibria with exchange rates on the order of milliseconds, the kinetic parameters for Φ-value analysis can be obtained from NMR relaxation dispersion experiments, under fully native conditions, along with a wealth of high-resolution structural information about the states in exchange (native, denatured, and intermediate states that populate the pathway). This additional structural information, which is not readily obtained through stopped-flow based methods, can significantly facilitate the interpretation of Φ values because it often reports on the validity of the assumptions required for a rigorous interpretation of Φ values.

scholarly journals Mutational Analysis of the Putative Anti-Müllerian Hormone (AMH) Binding Interface on its Type II Receptor, AMHR2

Endocrinology ◽  
2020 ◽  
Vol 161 (7) ◽  
Author(s):  
Kaitlin N Hart ◽  
David Pépin ◽  
Magdalena Czepnik ◽  
Patricia K Donahoe ◽  
Thomas B Thompson
Keyword(s):  
Ligand Binding ◽  
Mutational Analysis ◽  
Structural Information ◽  
Luciferase Assay ◽  
Type Ii ◽  
Native Page ◽  
Binding Interface ◽  
Receptor Interactions ◽  
Development And Differentiation ◽  
Responsive Cell

Abstract Anti-Müllerian hormone (AMH) or Müllerian inhibiting substance is a unique member of the TGF-β family responsible for development and differentiation of the reproductive system. AMH signals through its own dedicated type II receptor, anti-Müllerian hormone receptor type II (AMHR2), providing an exclusive ligand-receptor pair within the broader TGF-β family. In this study, we used previous structural information to derive a model of AMH bound to AMHR2 to guide mutagenesis studies to identify receptor residues important for AMH signaling. Nonconserved mutations were introduced in AMHR2 and characterized in an AMH-responsive cell-based luciferase assay and native PAGE. Collectively, our results identified several residues important for AMH signaling within the putative ligand binding interface of AMHR2. Our results show that AMH engages AMHR2 at a similar interface to how activin and BMP class ligands bind the type II receptor, ACVR2B; however, there are significant molecular differences at the ligand interface of these 2 receptors, where ACVR2B is mostly hydrophobic and AMHR2 is predominately charged. Overall, this study shows that although the location of ligand binding on the receptor is similar to ACVR2A, ACVR2B, and BMPR2; AMHR2 uses unique ligand-receptor interactions to impart specificity for AMH.

Site-directed mutagenesis of an extradiol dioxygenase involved in tetralin biodegradation identifies residues important for activity or substrate specificity

Microbiology ◽  
2003 ◽  
Vol 149 (6) ◽  
pp. 1559-1567 ◽  
Author(s):  
Eloísa Andújar ◽  
Eduardo Santero
Keyword(s):  
Substrate Specificity ◽  
Binding Site ◽  
Mutational Analysis ◽  
Structural Information ◽  
Substrate Inhibition ◽  
Site Directed Mutagenesis ◽  
Functional Importance ◽  
Extradiol Dioxygenase ◽  
Polycyclic Compounds ◽  
Extradiol Dioxygenases

The sequence of the extradiol dioxygenase ThnC, involved in tetralin biodegradation, was aligned with other extradiol dioxygenases involved in biodegradation of polycyclic compounds, and a three-dimensional model of ThnC, based on the structure of the previously crystallized 2,3-dihydroxybiphenyl dioxygenase from Burkholderia fungorum LB400, was built. In order to assess the functional importance of some non-active-site residues whose relevance could not be established by structural information, a number of positions surrounding the substrate-binding site were mutated in ThnC. Ten mutant proteins were purified and their activity towards 1,2-dihydroxytetralin, 1,2-dihydroxynaphthalene and 2,3-dihydroxybiphenyl was characterized. N213H, Q198H, G206M, A282R and A282G mutants increased k cat/K m at least twofold using 1,2-dihydroxytetralin as the substrate, thus showing that activity of ThnC is not maximized for this substrate. N213H and Q198H mutants increased k cat/K m using any of the substrates tested, thus showing the relevance for activity of these two histidines, which are highly conserved in dihydroxybiphenyl dioxygenases, but not present in dihydroxynaphthalene dioxygenases. Different substitutions in position 282 had different effects on general activity or substrate specificity, thus showing the functional importance of the most C-terminal β-sheet of the protein. A251M and G206M mutants showed increased activity specifically for a particular substrate. N213H, G206M, A282R, A282G and Y177I substitutions resulted in enzymes more tolerant to acidic pH, the most striking effect being observed in mutant Y177I, which showed maximal activity at pH 5·5. In addition, Q198D and V175D mutants, which had altered K m, also showed altered sensitivity to substrate inhibition, thus indicating that inhibition is exerted through the same binding site. This mutational analysis, therefore, identified conserved residues important for activity or substrate specificity, and also shed some light on the mechanism of substrate inhibition exhibited by extradiol dioxygenases.

Mutational analysis ofH3F3A, a chromatin remodeling gene in common solid tumors

Apmis ◽  
2013 ◽  
Vol 122 (1) ◽  
pp. 81-82
Author(s):  
Eun Mi Je ◽  
Nam Jin Yoo ◽  
Sug Hyung Lee
Keyword(s):  
Solid Tumors ◽  
Chromatin Remodeling ◽  
Mutational Analysis

Mutational Analysis of the BPTI Folding Pathway

1995 ◽  
pp. 483-492
Author(s):  
David P. Goldenberg ◽  
Jose A. Mendoza ◽  
Jian-Xin Zhang
Keyword(s):  
Mutational Analysis ◽  
Folding Pathway

Rigid backbone moiety of KNI-272, a highly selective HIV protease inhibitor: methanol, acetone and dimethylsulfoxide solvated forms of 3-[3-benzyl-2-hydroxy-9-(isoquinolin-5-yloxy)-6-methylsulfanylmethyl-5,8-dioxo-4,7-diazanonanoyl]-N-tert-butyl-1,3-thiazolidine-4-carboxamide

2004 ◽  
Vol 60 (4) ◽  
pp. 433-437 ◽  
Author(s):  
Mitsunobu Doi ◽  
Tooru Kimura ◽  
Toshimasa Ishida ◽  
Yoshiaki Kiso
Keyword(s):  
Transition State ◽  
Protease Inhibitor ◽  
Terminal Region ◽  
Hiv Protease ◽  
Hiv Protease Inhibitor ◽  
Remarkable Similarity ◽  
Conformational Variations ◽  
Solvent Systems ◽  
Local Conformations ◽  
Phenylbutyric Acid

When crystals of kynostatin (KNI)-272, a highly selective HIV protease inhibitor containing allophenylnorstatine [(2S,3S)-3-amino-2-hydroxy-4-phenylbutyric acid], were grown in three different solvent systems (methanol, acetone and dimethylsulfoxide solutions), the local conformations around the hydroxymethylcarbonyl (HMC) moiety, which mimics the structure of the transition state, were similar in all three forms. The peptide backbones were slightly bent, but their structures differed from typical sheets, turns or helixes. Although the isoquinoline ring at the N-terminal showed conformational variations, a remarkable similarity was observed in the C-terminal region, including the HMC moiety. Moreover, the conformational characteristics of the uncomplexed forms resembled those of the inhibitor within the KNI-272–HIV protease complex. This suggests that the structure of the C-terminal region of KNI-272 is rigid or very stable.

scholarly journals Mutational Analysis of Transmembrane Regions 3 and 4 of SecY, a Central Component of Protein Translocase

2004 ◽  
Vol 186 (12) ◽  
pp. 3960-3969 ◽  
Author(s):  
Hiroyuki Mori ◽  
Naomi Shimokawa ◽  
Yasunari Satoh ◽  
Koreaki Ito
Keyword(s):  
Mutational Analysis ◽  
Protein Translocation ◽  
Structural Information ◽  
Synthetic Lethality ◽  
Contact Point ◽  
Alpha Helix ◽  
Growth Defect ◽  
Central Component ◽  
Contact Points ◽  
Specific Contact

ABSTRACT The SecYEG heterotrimeric membrane protein complex functions as a channel for protein translocation across the Escherichia coli cytoplasmic membrane. SecY is the central subunit of the SecYEG complex and contains 10 transmembrane segments (TM1 to TM10). Previous mutation studies suggested that TM3 and TM4 are particularly important for SecY function. To further characterize TM3 and TM4, we introduced a series of cysteine-scanning mutations into these segments. With one exception (an unstable product), all the mutant proteins complemented the cold-sensitive growth defect of the secY39 mutant. A combination of this secY mutation and the secG deletion resulted in synthetic lethality, and the TM3 and TM4 SecY cysteine substitution mutations were examined for their ability to complement this lethality. Although they were all positive for complementation, some of the complemented cells exhibited significant retardation of protein export. The substitution-sensitive residues in TM3 can be aligned to one side of the alpha-helix, and those in TM4 revealed a tendency for residues closer to the cytosolic side of the membrane to be more severely affected. Disulfide cross-linking experiments identified a specific contact point for TM3 and SecG TM2 as well as for TM4 and SecG TM1. Thus, although TM3 and TM4 do not contain any single residue that is absolutely required, they include functionally important helix surfaces and specific contact points with SecG. These results are discussed in light of the structural information available for the SecY complex.

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