scholarly journals Structure Determination of Inactive-State GPCRs with a Universal Nanobody

2021 ◽  
Author(s):  
Michael J. Robertson ◽  
Feng He ◽  
Justin G. Meyerowitz ◽  
Alpay B. Seven ◽  
Ouliana Panova ◽  
...  
Keyword(s):  
Somatostatin Receptor ◽  
Protein Structures ◽  
Single Chain ◽  
Intracellular Loop ◽  
Single Chain Antibody ◽  
Inactive State ◽  
X Ray Crystallography ◽  
High Resolution Map ◽  
Μ Opioid Receptor

Cryogenic electron microscopy (cryo-EM) has widened the field of structure-based drug discovery by allowing for routine determination of membrane protein structures previously intractable. However, despite representing one of the largest classes of therapeutic targets, most inactive-state G protein-coupled receptors (GPCRs) have remained inaccessible for cryo-EM because their small size and membrane-embedded nature impedes projection alignment for high-resolution map reconstructions. Here we demonstrate that a camelid single-chain antibody (nanobody) recognizing a grafted intracellular loop can be used to obtain cryo-EM structures of different inactive-state GPCRs at resolutions comparable or better than those obtained by X-ray crystallography. Using this approach, we obtained the structure of human neurotensin 1 receptor (NTSR1) bound to antagonist SR48692, of μ-opioid receptor (MOR) bound to the clinical antagonist alvimopan, as well as the structure of the previously uncharacterized somatostatin receptor 2 (SSTR2) in the apo state; each of these structures yields novel insights into ligand binding and specificity. We expect this rapid, straightforward approach to facilitate the broad structural exploration of GPCR inactive states without the need for extensive engineering and crystallization.

scholarly journals Role of Computational Methods in Going beyond X-ray Crystallography to Explore Protein Structure and Dynamics

2018 ◽  
Vol 19 (11) ◽  
pp. 3401 ◽  
Author(s):  
Ashutosh Srivastava ◽  
Tetsuro Nagai ◽  
Arpita Srivastava ◽  
Osamu Miyashita ◽  
Florence Tama
Keyword(s):  
Protein Dynamics ◽  
Computational Methods ◽  
Protein Structures ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
X Ray ◽  
X Ray Crystallography ◽  
Insight Into

Protein structural biology came a long way since the determination of the first three-dimensional structure of myoglobin about six decades ago. Across this period, X-ray crystallography was the most important experimental method for gaining atomic-resolution insight into protein structures. However, as the role of dynamics gained importance in the function of proteins, the limitations of X-ray crystallography in not being able to capture dynamics came to the forefront. Computational methods proved to be immensely successful in understanding protein dynamics in solution, and they continue to improve in terms of both the scale and the types of systems that can be studied. In this review, we briefly discuss the limitations of X-ray crystallography in studying protein dynamics, and then provide an overview of different computational methods that are instrumental in understanding the dynamics of proteins and biomacromolecular complexes.

scholarly journals "From one seed a whole handful": homologous proteins as seeds in crystallisation

2014 ◽  
Vol 70 (a1) ◽  
pp. C1144-C1144
Author(s):  
Areej Abuhammad ◽  
Michael McDonough ◽  
Jürgen Brem ◽  
Christopher Schofield ◽  
Elspeth Garman
Keyword(s):  
Protein Crystallization ◽  
Protein Structures ◽  
Crystallization Time ◽  
Homologous Proteins ◽  
X Ray Crystallography ◽  
Alternative Approach ◽  
Crystallization Conditions ◽  
The Right

Protein structures have significantly impacted and aided drug discovery efforts. However, it is not enough to know the structure of a protein; it must be the right structure. Small alteration in sequence can lead to different conformations and oligomerization states, cause changes which lead to different active site architecture and also which modify function. Protein crystallization is an essential prerequisite for the determination of protein structures by X-ray crystallography. We have obtained encouraging initial results for a hitherto unexplored crystallization method with the enzyme arylamine N-acetyltransferase from M. tuberculosis (TBNAT). Despite prolonged and varied trials to crystallize TBNAT, an important anti-tubercular drug target, no crystals were obtained. In an alternative approach, cross-seeding of TBNAT protein with micro-crystalline seeds from a homologous NAT from M. marinum (74 % sequence identity (SID)) surprisingly resulted in a single 20 micron sized TBNAT crystal that diffracted to 2.1 Å and allowed for TBNAT structure determination (Abuhammad et al., 2013). To our knowledge, cross-seeding crystallisation using homologous proteins has only been previously successful in cases with more than 85% SID. In this study, we have explored the effect of low sequence homology on cross seeding using β-lactamases with SID as low as 30%. Despite the low SIDs, the results show cross seeding leads to an increase in hits obtained, the identification of new crystallization conditions, shortening of crystallization time and an improvement in the quality of the crystals obtained.

Membrane protein crystallography in the era of modern structural biology

2020 ◽  
Vol 48 (6) ◽  
pp. 2505-2524
Author(s):  
Tristan O. C. Kwan ◽  
Danny Axford ◽  
Isabel Moraes
Keyword(s):  
Structural Biology ◽  
Molecular Level ◽  
Protein Structures ◽  
Three Dimensional ◽  
Biological Macromolecules ◽  
X Ray ◽  
X Ray Crystallography ◽  
Cellular Processes ◽  
Biochemical Level

The aim of structural biology has been always the study of biological macromolecules structures and their mechanistic behaviour at molecular level. To achieve its goal, multiple biophysical methods and approaches have become part of the structural biology toolbox. Considered as one of the pillars of structural biology, X-ray crystallography has been the most successful method for solving three-dimensional protein structures at atomic level to date. It is however limited by the success in obtaining well-ordered protein crystals that diffract at high resolution. This is especially true for challenging targets such as membrane proteins (MPs). Understanding structure-function relationships of MPs at the biochemical level is vital for medicine and drug discovery as they play critical roles in many cellular processes. Though difficult, structure determination of MPs by X-ray crystallography has significantly improved in the last two decades, mainly due to many relevant technological and methodological developments. Today, numerous MP crystal structures have been solved, revealing many of their mechanisms of action. Yet the field of structural biology has also been through significant technological breakthroughs in recent years, particularly in the fields of single particle electron microscopy (cryo-EM) and X-ray free electron lasers (XFELs). Here we summarise the most important advancements in the field of MP crystallography and the significance of these developments in the present era of modern structural biology.

The immunoreactivity of the anti-p21Ras single-chain fragment variant KGH-R1 and its predicted binding sites to p21Ras

Immunotherapy ◽  
2020 ◽  
Vol 12 (12) ◽  
pp. 879-890
Author(s):  
Peng Wang ◽  
Xinyan Pan ◽  
Qiang Feng ◽  
Hong Zou ◽  
Jing Cui ◽  
...  
Keyword(s):  
Binding Sites ◽  
Protein Structures ◽  
Antibody Fragment ◽  
Human Tumor ◽  
Wild Type ◽  
Single Chain ◽  
Single Chain Antibody ◽  
Chain Fragment ◽  
Complementarity Determining Regions ◽  
Single Chain Fragment

Aim: Previously, we constructed a novel anti-p21Ras single-chain antibody fragment, KGH-R1-single-chain fragment variant (ScFv). However, the immunoreactivity of this antibody toward p21Ras is still unclear. Materials & methods: ELISAs, immunohistochemistry, western blotting and immunofluorescence were used to identify the immunoreactivity of KGH-R1-ScFv toward p21Ras. An in silico approach was used to determine the protein structures of KGH-R1-ScFv and p21Ras and then to predict the site involved in the binding of KGH-R1-ScFv to p21Ras. Results: KGH-R1-ScFv had a specific immune reaction with prokaryotically expressed p21Ras, human tumor cells and tumor tissues with RAS mutations or overexpression of RAS. Molecular docking showed that KGH-R1-ScFv could stably interact with wild-type and mutant p21Ras and the binding sites were in the complementarity-determining regions of KGH-R1-ScFv. Conclusion: KGH-R1-ScFv shows specific immunoreactivity toward wild-type and mutant p21Ras as well as the corresponding tumors, which suggests that KGH-R1-ScFv shows potential as a therapeutic antibody for therapy of RAS-driven tumors.

Determination of active single chain antibody concentrations in crude periplasmic fractions

1996 ◽  
Vol 194 (2) ◽  
pp. 201-209 ◽  
Author(s):  
Bert Kazemier ◽  
de Haard Hans ◽  
Piet Boender ◽  
Bob van Gemen ◽  
Hennie Hoogenboom
Keyword(s):  
Single Chain ◽  
Single Chain Antibody

scholarly journals Introducing site-specific cysteines into nanobodies for mercury labelling allowsde novophasing of their crystal structures

2017 ◽  
Vol 73 (10) ◽  
pp. 804-813 ◽  
Author(s):  
Simon Boje Hansen ◽  
Nick Stub Laursen ◽  
Gregers Rom Andersen ◽  
Kasper R. Andersen
Keyword(s):  
Structure Determination ◽  
De Novo ◽  
Protein Complexes ◽  
Protein Structures ◽  
Phase Information ◽  
X Ray Crystallography ◽  
Isomorphous Replacement ◽  
High Quality Protein ◽  
Radiation Induced

The generation of high-quality protein crystals and the loss of phase information during an X-ray crystallography diffraction experiment represent the major bottlenecks in the determination of novel protein structures. A generic method for introducing Hg atoms into any crystal independent of the presence of free cysteines in the target protein could considerably facilitate the process of obtaining unbiased experimental phases. Nanobodies (single-domain antibodies) have recently been shown to promote the crystallization and structure determination of flexible proteins and complexes. To extend the usability of nanobodies for crystallographic work, variants of the Nb36 nanobody with a single free cysteine at one of four framework-residue positions were developed. These cysteines could be labelled with fluorophores or Hg. For one cysteine variant (Nb36-C85) two nanobody structures were experimentally phased using single-wavelength anomalous dispersion (SAD) and single isomorphous replacement with anomalous signal (SIRAS), taking advantage of radiation-induced changes in Cys–Hg bonding. Importantly, Hg labelling influenced neither the interaction of Nb36 with its antigen complement C5 nor its structure. The results suggest that Cys–Hg-labelled nanobodies may become efficient tools for obtainingde novophase information during the structure determination of nanobody–protein complexes.

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