New structural insights into anomeric carbohydrate recognition by frutalin: an α-d-galactose-binding lectin from breadfruit seeds

2019 ◽  
Vol 476 (1) ◽  
pp. 101-113 ◽  
Author(s):  
Antonio Eufrásio Vieira Neto ◽  
Felipe Domingos de Sousa ◽  
Humberto D'Muniz Pereira ◽  
Frederico Bruno Mendes Batista Moreno ◽  
Marcos Roberto Lourenzoni ◽  
...  
Keyword(s):  
Amino Acids ◽  
Biological Activities ◽  
3D Structure ◽  
Carbohydrate Binding ◽  
X Ray Crystallography ◽  
Multiple Binding Sites ◽  
Multiple Binding ◽  
New Perspective ◽  
Dynamics Simulations ◽  
Binding Lectin

Abstract Frutalin (FTL) is a multiple-binding lectin belonging to the jacalin-related lectin (JRL) family and derived from Artocarpus incisa (breadfruit) seeds. This lectin specifically recognizes and binds α-d-galactose. FTL has been successfully used in immunobiological research for the recognition of cancer-associated oligosaccharides. However, the molecular bases by which FTL promotes these specific activities remain poorly understood. Here, we report the whole 3D structure of FTL for the first time, as determined by X-ray crystallography. The obtained crystals diffracted to 1.81 Å (Apo-frutalin) and 1.65 Å (frutalin–d-Gal complex) of resolution. The lectin exhibits post-translational cleavage yielding an α- (133 amino acids) and β-chain (20 amino acids), presenting a homotetramer when in solution, with a typical JRL β-prism. The β-prism was composed of three 4-stranded β-sheets forming three antiparallel Greek key motifs. The carbohydrate-binding site (CBS) involved the N-terminus of the α-chain and was formed by four key residues: Gly25, Tyr146, Trp147 and Asp149. Together, these results were used in molecular dynamics simulations in aqueous solutions to shed light on the molecular basis of FTL-ligand binding. The simulations suggest that Thr-Ser-Ser-Asn (TSSN) peptide excision reduces the rigidity of the FTL CBS, increasing the number of interactions with ligands and resulting in multiple-binding sites and anomeric recognition of α-d-galactose sugar moieties. Our findings provide a new perspective to further elucidate the versatility of FTL in many biological activities.

scholarly journals Moving toward generalizable NZ-1 labeling for 3D structure determination with optimized epitope-tag insertion

2021 ◽  
Vol 77 (5) ◽  
pp. 645-662
Author(s):  
Risako Tamura-Sakaguchi ◽  
Rie Aruga ◽  
Mika Hirose ◽  
Toru Ekimoto ◽  
Takuya Miyake ◽  
...  
Keyword(s):  
Complex Formation ◽  
Structure Determination ◽  
Structural Changes ◽  
Insertion Site ◽  
3D Structure ◽  
Binding Pocket ◽  
Crystallographic Analysis ◽  
Stable Complex ◽  
X Ray Crystallography ◽  
Dynamics Simulations

Antibody labeling has been conducted extensively for structure determination using both X-ray crystallography and electron microscopy (EM). However, establishing target-specific antibodies is a prerequisite for applying antibody-assisted structural analysis. To expand the applicability of this strategy, an alternative method has been developed to prepare an antibody complex by inserting an exogenous epitope into the target. It has already been demonstrated that the Fab of the NZ-1 monoclonal antibody can form a stable complex with a target containing a PA12 tag as an inserted epitope. Nevertheless, it was also found that complex formation through the inserted PA12 tag inevitably caused structural changes around the insertion site on the target. Here, an attempt was made to improve the tag-insertion method, and it was consequently discovered that an alternate tag (PA14) could replace various loops on the target without inducing large structural changes. Crystallographic analysis demonstrated that the inserted PA14 tag adopts a loop-like conformation with closed ends in the antigen-binding pocket of the NZ-1 Fab. Due to proximity of the termini in the bound conformation, the more optimal PA14 tag had only a minor impact on the target structure. In fact, the PA14 tag could also be inserted into a sterically hindered loop for labeling. Molecular-dynamics simulations also showed a rigid structure for the target regardless of PA14 insertion and complex formation with the NZ-1 Fab. Using this improved labeling technique, negative-stain EM was performed on a bacterial site-2 protease, which enabled an approximation of the domain arrangement based on the docking mode of the NZ-1 Fab.

scholarly journals Moving toward generalizable NZ-1 labeling for 3D structure determination with optimized epitope tag insertion

2020 ◽  
Author(s):  
Risako Tamura-Sakaguchi ◽  
Rie Aruga ◽  
Mika Hirose ◽  
Toru Ekimoto ◽  
Takuya Miyake ◽  
...  
Keyword(s):  
Structural Change ◽  
Complex Formation ◽  
Structure Determination ◽  
Insertion Site ◽  
3D Structure ◽  
Binding Pocket ◽  
Stable Complex ◽  
X Ray Crystallography ◽  
Antibody Complex ◽  
Dynamics Simulations

AbstractAntibody labeling has been extensively conducted for structure determination in both x-ray crystallography and EM analysis. However, establishing target-specific antibodies is a prerequisite for applying antibody-assisted structural analysis. To expand the applicability of this strategy, we have developed an alternative method to prepare an antibody-complex by inserting an exogenous epitope into the target. We have already demonstrated that the Fab of monoclonal antibody NZ-1 could form a stable complex with the target containing a PA12 tag as an inserted epitope. Nevertheless, we also found that the complex formation through the inserted PA12 tag inevitably caused structural change around the insertion site of the target. Hence, we here attempted to improve the insertion method and consequently discovered that utilization of a PA14 tag significantly reduced the structural change in the target. By adopting a closed ring-like conformation inside the antigen-binding pocket, the inserted PA14 tag had less impact on the folding of the target. Due to this structural property, the PA14 tag could also be inserted into the sterically hindered loop for labeling. Molecular dynamics simulations also indicated that the folding of the target was rigid regardless of the PA14 insertion and the complex formation with the NZ-1 Fab. Using the improved labeling technique, we performed negative-stain EM on a bacterial site-2 protease, which enabled us to approximate the domain arrangement based on the docking mode of the NZ-1 Fab.

scholarly journals 3D Structural Fluctuation of IgG1 Antibody Revealed by Individual Particle Electron Tomography

2015 ◽  
Vol 5 (1) ◽  
Author(s):  
Xing Zhang ◽  
Lei Zhang ◽  
Huimin Tong ◽  
Bo Peng ◽  
Matthew J. Rames ◽  
...  
Keyword(s):  
Protein Dynamics ◽  
Electron Tomography ◽  
3D Structure ◽  
Three Dimensional ◽  
Individual Particle ◽  
Single Particle Reconstruction ◽  
X Ray Crystallography ◽  
Igg1 Antibody ◽  
Structural Fluctuation ◽  
Dynamics Simulations

Abstract Commonly used methods for determining protein structure, including X-ray crystallography and single-particle reconstruction, often provide a single and unique three-dimensional (3D) structure. However, in these methods, the protein dynamics and flexibility/fluctuation remain mostly unknown. Here, we utilized advances in electron tomography (ET) to study the antibody flexibility and fluctuation through structural determination of individual antibody particles rather than averaging multiple antibody particles together. Through individual-particle electron tomography (IPET) 3D reconstruction from negatively-stained ET images, we obtained 120 ab-initio 3D density maps at an intermediate resolution (~1–3 nm) from 120 individual IgG1 antibody particles. Using these maps as a constraint, we derived 120 conformations of the antibody via structural flexible docking of the crystal structure to these maps by targeted molecular dynamics simulations. Statistical analysis of the various conformations disclosed the antibody 3D conformational flexibility through the distribution of its domain distances and orientations. This blueprint approach, if extended to other flexible proteins, may serve as a useful methodology towards understanding protein dynamics and functions.

scholarly journals Novel quaternary structures of the human prion protein globular domain

2020 ◽  
Author(s):  
Leandro Oliveira Bortot ◽  
Victor Lopes Rangel ◽  
Francesca A. Pavlovici ◽  
Kamel El Omari ◽  
Armin Wagner ◽  
...  
Keyword(s):  
Drug Discovery ◽  
Prion Protein ◽  
3D Structure ◽  
Disulfide Bridge ◽  
Cellular Prion Protein ◽  
Crystallographic Structure ◽  
Globular Domain ◽  
X Ray Crystallography ◽  
Quaternary Structures ◽  
Dynamics Simulations

AbstractPrion disease is caused by the misfolding of the cellular prion protein, PrPC, into a self-templating conformer, PrPSc. Nuclear magnetic resonance (NMR) and X-ray crystallography revealed the 3D structure of the globular domain of PrPC and the possibility of its dimerization via an interchain disulfide bridge that forms due to domain swap or by non-covalent association of two monomers. On the contrary, PrPSc is composed by a complex and heterogeneous ensemble of poorly defined conformations and quaternary arrangements that are related to different patterns of neurotoxicity. Targeting PrPC with molecules that stabilize the native conformation of its globular domain emerged as a promising approach to develop anti-prion therapies. One of the advantages of this approach is employing structure-based drug discovery methods to PrPC. Thus, it is essential to expand our structural knowledge about PrPC as much as possible to aid such drug discovery efforts. In this work, we report a crystallographic structure of the globular domain of human PrPC that shows a novel dimeric form and a novel oligomeric arrangement. We use molecular dynamics simulations to explore its structural dynamics and stability and discuss potential implications of these new quaternary structures to the conversion process.

Hierarchy of π-Stacking Determines the Conformational Preference of Bis-Squaraines

2020 ◽  
Author(s):  
Abhishek Singh ◽  
Reman K. Singh ◽  
G Naresh Patwari
Keyword(s):  
Hydrogen Bonding ◽  
Rational Design ◽  
Biological Molecules ◽  
Conformational Space ◽  
X Ray Crystallography ◽  
Simple Strategy ◽  
Induced Folding ◽  
Π Stacking ◽  
Varying Length ◽  
Dynamics Simulations

The rational design of conformationally controlled foldable modules can lead to a deeper insight into the conformational space of complex biological molecules where non-covalent interactions such as hydrogen bonding and π-stacking are known to play a pivotal role. Squaramides are known to have excellent hydrogen bonding capabilities and hence, are ideal molecules for designing foldable modules that can mimic the secondary structures of bio-molecules. The π-stacking induced folding of bis-squaraines tethered using aliphatic primary and secondary-diamine linkers of varying length is explored with a simple strategy of invoking small perturbations involving the length linkers and degree of substitution. Solution phase NMR investigations in combination with molecular dynamics simulations suggest that bis-squaraines predominantly exist as extended conformations. Structures elucidated by X-ray crystallography confirmed a variety of folded and extended secondary conformations including hairpin turns and 𝛽-sheets which are determined by the hierarchy of π-stacking relative to N–H···O hydrogen bonds.

Hierarchy of π-Stacking Determines the Conformational Preference of Bis-Squaraines

2020 ◽  
Author(s):  
Abhishek Singh ◽  
Reman K. Singh ◽  
G Naresh Patwari
Keyword(s):  
Hydrogen Bonding ◽  
Rational Design ◽  
Biological Molecules ◽  
Conformational Space ◽  
X Ray Crystallography ◽  
Simple Strategy ◽  
Induced Folding ◽  
Π Stacking ◽  
Varying Length ◽  
Dynamics Simulations

The rational design of conformationally controlled foldable modules can lead to a deeper insight into the conformational space of complex biological molecules where non-covalent interactions such as hydrogen bonding and π-stacking are known to play a pivotal role. Squaramides are known to have excellent hydrogen bonding capabilities and hence, are ideal molecules for designing foldable modules that can mimic the secondary structures of bio-molecules. The π-stacking induced folding of bis-squaraines tethered using aliphatic primary and secondary-diamine linkers of varying length is explored with a simple strategy of invoking small perturbations involving the length linkers and degree of substitution. Solution phase NMR investigations in combination with molecular dynamics simulations suggest that bis-squaraines predominantly exist as extended conformations. Structures elucidated by X-ray crystallography confirmed a variety of folded and extended secondary conformations including hairpin turns and 𝛽-sheets which are determined by the hierarchy of π-stacking relative to N–H···O hydrogen bonds.

NMDAR PAMs: Multiple Chemotypes for Multiple Binding Sites

2019 ◽  
Vol 19 (24) ◽  
pp. 2239-2253 ◽  
Author(s):  
Paul J. Goldsmith
Keyword(s):  
Binding Sites ◽  
Receptor Activation ◽  
Research Area ◽  
Allosteric Modulators ◽  
Nonselective Cation Channels ◽  
Multiple Binding Sites ◽  
Multiple Binding ◽  
Excitatory Neurotransmission ◽  
Psychiatric Conditions ◽  
High Level

The N-methyl-D-aspartate receptor (NMDAR) is a member of the ionotropic glutamate receptor (iGluR) family that plays a crucial role in brain signalling and development. NMDARs are nonselective cation channels that are involved with the propagation of excitatory neurotransmission signals with important effects on synaptic plasticity. NMDARs are functionally and structurally complex receptors, they exist as a family of subtypes each with its own unique pharmacological properties. Their implication in a variety of neurological and psychiatric conditions means they have been a focus of research for many decades. Disruption of NMDAR-related signalling is known to adversely affect higherorder cognitive functions (e.g. learning and memory) and the search for molecules that can recover (or even enhance) receptor output is a current strategy for CNS drug discovery. A number of positive allosteric modulators (PAMs) that specifically attempt to overcome NMDAR hypofunction have been discovered. They include various chemotypes that have been found to bind to several different binding sites within the receptor. The heterogeneity of chemotype, binding site and NMDAR subtype provide a broad landscape of ongoing opportunities to uncover new features of NMDAR pharmacology. Research on NMDARs continues to provide novel mechanistic insights into receptor activation and this review will provide a high-level overview of the research area and discuss the various chemical classes of PAMs discovered so far.

Sign in / Sign up

Export Citation Format

Share Document