scholarly journals Copper(I)-mediated protein–protein interactions result from suboptimal interaction surfaces

2009 ◽  
Vol 422 (1) ◽  
pp. 37-42 ◽  
Author(s):  
Lucia Banci ◽  
Ivano Bertini ◽  
Vito Calderone ◽  
Nunzia Della-Malva ◽  
Isabella C. Felli ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Metal Binding ◽  
Site Directed Mutagenesis ◽  
Protein Protein Interactions ◽  
X Ray ◽  
X Ray Crystallography ◽  
Metal Binding Domain ◽  
Modelling Techniques ◽  
Cellular Pathways ◽  
P Type

The homoeostasis of metal ions in cells is the result of the contribution of several cellular pathways that involve transient, often weak, protein–protein interactions. Metal transfer typically implies the formation of adducts where the metal itself acts as a bridge between proteins, by co-ordinating residues of both interacting partners. In the present study we address the interaction between the human copper(I)-chaperone HAH1 (human ATX1 homologue) and a metal-binding domain in one of its partners, namely the P-type copper-transporting ATPase, ATP7A (ATPase, Cu+ transporting, α polypeptide). The adduct was structurally characterized in solution, in the presence of copper(I), and through X-ray crystallography, upon replacing copper(I) with cadmium(II). Further insight was obtained through molecular modelling techniques and site-directed mutagenesis. It was found that the interaction involves a relatively small interface (less than 1000 Å2, 1 Å=0.1 nm) with a low fraction of non-polar atoms. These observations provide a possible explanation for the low affinity of the two apoproteins. It appears that electrostatics is important in selecting which domain of the ATPase is able to form detectable amounts of the metal-mediated adduct with HAH1.

Regulative interactions of the osmosensing C-terminal domain in the trimeric glycine betaine transporter BetP from Corynebacterium glutamicum

2009 ◽  
Vol 390 (8) ◽  
Author(s):  
Reinhard Krämer ◽  
Christine Ziegler
Keyword(s):  
Amino Acid ◽  
Corynebacterium Glutamicum ◽  
Protein Interactions ◽  
Regulation Mechanism ◽  
Protein Protein Interactions ◽  
Molecular Switching ◽  
X Ray ◽  
X Ray Crystallography ◽  
Terminal Domain ◽  
Domain Reorientation

Abstract Activation of the osmoregulated trimeric betaine transporter BetP from Corynebacterium glutamicum was shown to depend mainly on the correct folding and integrity of its 55 amino acid long, partly α-helical C-terminal domain. Reorientation of the three C-terminal domains in the BetP trimer indicates different lipid-protein and protein-protein interactions of the C-terminal domain during osmoregulation. A regulation mechanism is suggested where this domain switches the transporter from the inactive to the active state. Interpretation of recently obtained electron and X-ray crystallography data of BetP led to a structure-function based model of C-terminal molecular switching involved in osmoregulation.

scholarly journals The C-terminal tail of the bacterial translocation ATPase SecA modulates its activity

2018 ◽  
Author(s):  
Mohammed Jamshad ◽  
Timothy J. Knowles ◽  
Scott A. White ◽  
Douglas G. Ward ◽  
Fiyaz Mohammed ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Metal Binding ◽  
Cytoplasmic Membrane ◽  
Protein Recognition ◽  
X Ray ◽  
Ribosome Binding ◽  
X Ray Crystallography ◽  
Substrate Protein ◽  
Metal Binding Domain

AbstractIn bacteria, the translocation of a subset of proteins across the cytoplasmic membrane by the Sec machinery requires SecA. Although SecA can recognise nascent polypeptides, the mechanism of cotranslational substrate protein recognition is not known. Here, we investigated the role of the C-terminal tail (CTT) of SecA, which consists of a flexible linker (FLD) and a small metal-binding domain (MBD), in its interaction with nascent polypeptides. Phylogenetic analysis and ribosome binding experiments indicated that the MBD interacts with 70S ribosomes. Disruption of the entire CTT or the MBD alone had opposing effects on ribosome binding, substrate-protein binding, ATPase activity and in vivo function. Autophotocrosslinking, mass spectrometry, x-ray crystallography and small-angle x-ray scattering experiments provided insight into the CTT-mediated conformational changes in SecA. Finally, photocrosslinking experiments indicated that binding of SecA to substrate protein affected its interaction with the ribosome. Taken together, our results suggest a mechanism for substrate protein recognition.Impact StatementSecA is an evolutionarily conserved ATPase that is required for the translocation of a subset of proteins across the cytoplasmic membrane in bacteria. We investigated how SecA recognises its substrate proteins at the ribosome as they are still being synthesised (i.e. cotranslationally).

New perceptions of transcription factor properties from NMR

1998 ◽  
Vol 76 (2-3) ◽  
pp. 368-378 ◽  
Author(s):  
Stefan Bagby ◽  
Cheryl H Arrowsmith ◽  
Mitsuhiko Ikura
Keyword(s):  
Transcription Factor ◽  
Protein Interactions ◽  
Dna Interaction ◽  
Protein Protein Interactions ◽  
Nmr Studies ◽  
X Ray ◽  
X Ray Crystallography ◽  
Protein Protein Interaction ◽  
Structural Problems ◽  
Induced Folding

The complementarity of NMR and X-ray crystallography for biomacromolecular studies has been particularly evident in analysis of transcription factor structures and interactions. While X-ray crystallography can be used to tackle relatively complicated structural problems including multicomponent (three and higher) complexes, NMR studies have provided new insights into the nature of protein-DNA and protein-protein interactions that would be difficult to obtain by other biophysical methods. We describe herein some of the novel and important information recently derived from NMR studies of transcription factors.Key words: protein-DNA interaction, protein-protein interaction, induced folding, conformational fluctuations, transcriptional regulation.

Decoding Protein-protein Interactions: An Overview

2020 ◽  
Vol 20 (10) ◽  
pp. 855-882
Author(s):  
Olivia Slater ◽  
Bethany Miller ◽  
Maria Kontoyianni
Keyword(s):  
Drug Discovery ◽  
Protein Interactions ◽  
Drug Repurposing ◽  
Protein Docking ◽  
Target Space ◽  
Protein Protein Interactions ◽  
X Ray Crystallography ◽  
Protein Protein Interaction ◽  
Interaction Sites ◽  
Long Time

Drug discovery has focused on the paradigm “one drug, one target” for a long time. However, small molecules can act at multiple macromolecular targets, which serves as the basis for drug repurposing. In an effort to expand the target space, and given advances in X-ray crystallography, protein-protein interactions have become an emerging focus area of drug discovery enterprises. Proteins interact with other biomolecules and it is this intricate network of interactions that determines the behavior of the system and its biological processes. In this review, we briefly discuss networks in disease, followed by computational methods for protein-protein complex prediction. Computational methodologies and techniques employed towards objectives such as protein-protein docking, protein-protein interactions, and interface predictions are described extensively. Docking aims at producing a complex between proteins, while interface predictions identify a subset of residues on one protein that could interact with a partner, and protein-protein interaction sites address whether two proteins interact. In addition, approaches to predict hot spots and binding sites are presented along with a representative example of our internal project on the chemokine CXC receptor 3 B-isoform and predictive modeling with IP10 and PF4.

scholarly journals Homogeneously N-glycosylated proteins derived from the GlycoDelete HEK293 cell line enable diffraction-quality crystallogenesis

2020 ◽  
Vol 76 (12) ◽  
pp. 1244-1255
Author(s):  
Sandra Kozak ◽  
Yehudi Bloch ◽  
Steven De Munck ◽  
Aleksandra Mikula ◽  
Isabel Bento ◽  
...  
Keyword(s):  
Cell Line ◽  
Protein Interactions ◽  
Successful Implementation ◽  
Structural Studies ◽  
Protein Fragments ◽  
X Ray ◽  
X Ray Crystallography ◽  
Cryo Electron Microscopy ◽  
Acid Protein ◽  
Stimulating Factor

Structural studies of glycoproteins and their complexes provide critical insights into their roles in normal physiology and disease. Most glycoproteins contain N-linked glycosylation, a key post-translation modification that critically affects protein folding and stability and the binding kinetics underlying protein interactions. However, N-linked glycosylation is often an impediment to yielding homogeneous protein preparations for structure determination by X-ray crystallography or other methods. In particular, obtaining diffraction-quality crystals of such proteins and their complexes often requires modification of both the type of glycosylation patterns and their extent. Here, we demonstrate the benefits of producing target glycoproteins in the GlycoDelete human embryonic kidney 293 cell line that has been engineered to produce N-glycans as short glycan stumps comprising N-acetylglucosamine, galactose and sialic acid. Protein fragments of human Down syndrome cell-adhesion molecule and colony-stimulating factor 1 receptor were obtained from the GlycoDelete cell line for crystallization. The ensuing reduction in the extent and complexity of N-glycosylation in both protein molecules compared with alternative glycoengineering approaches enabled their productive deployment in structural studies by X-ray crystallography. Furthermore, a third successful implementation of the GlycoDelete technology focusing on murine IL-12B is shown to lead to N-glycosylation featuring an immature glycan in diffraction-quality crystals. It is proposed that the GlycoDelete cell line could serve as a valuable go-to option for the production of homogeneous glycoproteins and their complexes for structural studies by X-ray crystallography and cryo-electron microscopy.

scholarly journals A hybrid approach reveals the allosteric regulation of GTP cyclohydrolase I

2020 ◽  
Vol 117 (50) ◽  
pp. 31838-31849
Author(s):  
Rebecca Ebenhoch ◽  
Simone Prinz ◽  
Susann Kaltwasser ◽  
Deryck J. Mills ◽  
Robert Meinecke ◽  
...  
Keyword(s):  
Crystal Packing ◽  
Substrate Binding ◽  
Regulatory Protein ◽  
Allosteric Regulation ◽  
Hybrid Approach ◽  
Site Directed Mutagenesis ◽  
Gtp Cyclohydrolase I ◽  
Gtp Cyclohydrolase ◽  
X Ray ◽  
X Ray Crystallography

Guanosine triphosphate (GTP) cyclohydrolase I (GCH1) catalyzes the conversion of GTP to dihydroneopterin triphosphate (H2NTP), the initiating step in the biosynthesis of tetrahydrobiopterin (BH4). Besides other roles, BH4 functions as cofactor in neurotransmitter biosynthesis. The BH4 biosynthetic pathway and GCH1 have been identified as promising targets to treat pain disorders in patients. The function of mammalian GCH1s is regulated by a metabolic sensing mechanism involving a regulator protein, GCH1 feedback regulatory protein (GFRP). GFRP binds to GCH1 to form inhibited or activated complexes dependent on availability of cofactor ligands, BH4 and phenylalanine, respectively. We determined high-resolution structures of human GCH1−GFRP complexes by cryoelectron microscopy (cryo-EM). Cryo-EM revealed structural flexibility of specific and relevant surface lining loops, which previously was not detected by X-ray crystallography due to crystal packing effects. Further, we studied allosteric regulation of isolated GCH1 by X-ray crystallography. Using the combined structural information, we are able to obtain a comprehensive picture of the mechanism of allosteric regulation. Local rearrangements in the allosteric pocket upon BH4 binding result in drastic changes in the quaternary structure of the enzyme, leading to a more compact, tense form of the inhibited protein, and translocate to the active site, leading to an open, more flexible structure of its surroundings. Inhibition of the enzymatic activity is not a result of hindrance of substrate binding, but rather a consequence of accelerated substrate binding kinetics as shown by saturation transfer difference NMR (STD-NMR) and site-directed mutagenesis. We propose a dissociation rate controlled mechanism of allosteric, noncompetitive inhibition.

Address of the President Lord Todd at the anniversary meeting, 30 November 1976

1977 ◽  
Vol 352 (1671) ◽  
pp. 451-462
Keyword(s):  
Protein Interactions ◽  
Complex Analysis ◽  
Organic Molecules ◽  
Heavy Atom ◽  
Vitamin B ◽  
Protein Protein Interactions ◽  
X Ray Crystallography ◽  
Polypeptide Chains ◽  
Complex Organic

The Copley Medal is awarded to Professor Dorothy M. C. Hodgkin, O. M., F. R. S. Professor Dorothy Hodgkin is distinguished for her research on the structure of complex organic molecules by the method of X-ray crystallography. She was among the first to appreciate the importance of heavy-atom phase-determining methods and these she used to effect the first complete determination of the stereochemistry of a sterol derivative in her analysis of cholesteryl iodide. The same powerful method of analysis and in particular her extraordinary gift of being able to interpret correctly the complex, partially resolved and often misleading electron density patterns that are first obtained, have been responsible for her success in elucidating the structures of many other important natural products, especially penicillin and vitamin B 12 . This last is by far the most beautiful and complex analysis which has yet been completed in this field and it is of fundamental importance to chemical science. In recent years Professor Hodgkin’s main interest has been devoted to the structure of insulin, on which she has been working on and off since 1935. Carried out with characteristic precision, this work has become a mine of stereochemical information relating to contacts between polypeptide chains and is of great significance for our interpretation of protein-protein interactions.

Signals regulating trafficking of Menkes (MNK; ATP7A) copper-translocating P-type ATPase in polarized MDCK cells

2004 ◽  
Vol 287 (5) ◽  
pp. C1463-C1471 ◽  
Author(s):  
M. Greenough ◽  
L. Pase ◽  
I. Voskoboinik ◽  
M. J. Petris ◽  
A. Wilson O'Brien ◽  
...  
Keyword(s):  
Metal Binding ◽  
Laser Scanning ◽  
Mdck Cells ◽  
Laser Scanning Microscopy ◽  
Site Directed Mutagenesis ◽  
Confocal Laser ◽  
Molecular Evidence ◽  
Copper Absorption ◽  
P Type

The Menkes protein (MNK; ATP7A) functions as a transmembrane copper-translocating P-type ATPase and plays a vital role in systemic copper absorption in the gut and copper reabsorption in the kidney. Polarized epithelial cells such as Madin-Darby canine kidney (MDCK) cells are a physiologically relevant model for systemic copper absorption and reabsorption in vivo. In this study, cultured MDCK cells were used to characterize MNK trafficking and enabled the identification of signaling motifs required to target the protein to specific membranes. Using confocal laser scanning microscopy and surface biotinylation we demonstrate that MNK relocalizes from the Golgi to the basolateral (BL) membrane under elevated copper conditions. As previously shown in nonpolarized cells, the metal binding sites in the NH2-terminal domain of MNK were found to be required for copper-regulated trafficking from the Golgi to the plasma membrane. These data provide molecular evidence that is consistent with the presumed role of this protein in systemic copper absorption in the gut and reabsorption in the kidney. Using site-directed mutagenesis, we identified a dileucine motif proximal to the COOH terminus of MNK that was critical for correctly targeting the protein to the BL membrane and a putative PDZ target motif that was required for localization at the BL membrane in elevated copper.

scholarly journals Getting into position: the catalytic mechanisms of protein ubiquitylation

2004 ◽  
Vol 379 (3) ◽  
pp. 513-525 ◽  
Author(s):  
Lori A. PASSMORE ◽  
David BARFORD
Keyword(s):  
Protein Interactions ◽  
Regulatory Elements ◽  
Substrate Selection ◽  
Protein Protein Interactions ◽  
Different Types ◽  
Catalytic Mechanisms ◽  
Cellular Pathways ◽  
Ubiquitin Conjugating Enzymes ◽  
Insight Into

The role of protein ubiquitylation in the control of diverse cellular pathways has recently gained widespread attention. Ubiquitylation not only directs the targeted destruction of tagged proteins by the 26 S proteasome, but it also modulates protein activities, protein–protein interactions and subcellular localization. An understanding of the components involved in protein ubiquitylation (E1s, E2s and E3s) is essential to understand how specificity and regulation are conferred upon these pathways. Much of what we know about the catalytic mechanisms of protein ubiquitylation comes from structural studies of the proteins involved in this process. Indeed, structures of ubiquitin-activating enzymes (E1s) and ubiquitin-conjugating enzymes (E2s) have provided insight into their mechanistic details. E3s (ubiquitin ligases) contain most of the substrate specificity and regulatory elements required for protein ubiquitylation. Although several E3 structures are available, the specific mechanistic role of E3s is still unclear. This review will discuss the different types of ubiquitin signals and how they are generated. Recent advances in the field of protein ubiquitylation will be examined, including the mechanisms of E1, E2 and E3. In particular, we discuss the complexity of molecular recognition required to impose selectivity on substrate selection and topology of poly-ubiquitin chains.

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