scholarly journals Homology Modeling-Based in Silico Affinity Maturation Improves the Affinity of a Nanobody

2019 ◽  
Vol 20 (17) ◽  
pp. 4187 ◽  
Author(s):  
Xin Cheng ◽  
Jiewen Wang ◽  
Guangbo Kang ◽  
Min Hu ◽  
Bo Yuan ◽  
...  
Keyword(s):  
Homology Modeling ◽  
Rational Design ◽  
Structural Model ◽  
Three Dimensional ◽  
Phage Display Library ◽  
Affinity Maturation ◽  
New Strategy ◽  
Design Protocol ◽  
High Binding Affinity

Affinity maturation and rational design have a raised importance in the application of nanobody (VHH), and its unique structure guaranteed these processes quickly done in vitro. An anti-CD47 nanobody, Nb02, was screened via a synthetic phage display library with 278 nM of KD value. In this study, a new strategy based on homology modeling and Rational Mutation Hotspots Design Protocol (RMHDP) was presented for building a fast and efficient platform for nanobody affinity maturation. A three-dimensional analytical structural model of Nb02 was constructed and then docked with the antigen, the CD47 extracellular domain (CD47ext). Mutants with high binding affinity are predicted by the scoring of nanobody-antigen complexes based on molecular dynamics trajectories and simulation. Ultimately, an improved mutant with an 87.4-fold affinity (3.2 nM) and 7.36 °C higher thermal stability was obtained. These findings might contribute to computational affinity maturation of nanobodies via homology modeling using the recent advancements in computational power. The add-in of aromatic residues which formed aromatic-aromatic interaction plays a pivotal role in affinity and thermostability improvement. In a word, the methods used in this study might provide a reference for rapid and efficient in vitro affinity maturation of nanobodies.

scholarly journals Rational Design of a Triple-Layered Coaxial Extruder System: in silico and in vitro Evaluations Directed Towards Optimizing Cell Viability

2020 ◽  
Vol 6 (4) ◽  
Author(s):  
Christian Augusto Silva ◽  
Carlos J Cortés-Rodriguez ◽  
Jonas Hazur ◽  
Supachai Reakasame ◽  
Aldo R. Boccaccini
Keyword(s):  
Cell Viability ◽  
In Silico ◽  
Rational Design ◽  
Three Dimensional ◽  
Complex Structures ◽  
Vascular Networks ◽  
Coaxial Nozzle ◽  
Wide Availability ◽  
Cell Functionality

Biofabrication is a rapidly evolving field whose main goal is the manufacturing of three-dimensional (3D) cell-laden constructs that closely mimic tissues and organs. Despite recent advances on materials and techniques directed toward the achievement of this goal, several aspects such as tissue vascularization and prolonged cell functionality are limiting bench-to-bedside translation. Extrusion-based 3D bioprinting has been devised as a promising biofabrication technology to overcome these limitations, due to its versatility and wide availability. Here, we report the development of a triple-layered coaxial nozzle for use in the biomanufacturing of vascular networks and vessels. The design of the coaxial nozzle was first optimized toward guaranteeing high cell viability upon extrusion. This was done with the aid of in silico evaluations and their subsequent experimental validation by investigating the bioprinting of an alginate-based bioink. Results confirmed that the values for pressure distribution predicted by in silico experiments resulted in cell viabilities above 70% and further demonstrated the effect of layer thickness and extrusion pressure on cell viability. Our work paves the way for the rational design of multi-layered coaxial extrusion systems to be used in biofabrication approaches to replicate the very complex structures found in native organs and tissues.

scholarly journals Potent germline-like monoclonal antibodies: Rapid identification of promising candidates for antibody-based antiviral therapy

2021 ◽  
Author(s):  
Xiaoyi Zhu ◽  
Fei Yu ◽  
Yanling Wu ◽  
Tianlei Ying
Keyword(s):  
Monoclonal Antibodies ◽  
Rational Design ◽  
Large Scale ◽  
Molecular Mechanisms ◽  
Viral Infections ◽  
Somatic Hypermutation ◽  
Affinity Maturation ◽  
Human Mabs

Abstract Recent years, fully human monoclonal antibodies (mAbs) are making up an increasing share of the pharmaceutical market. However, to improve affinity and efficacy of antibodies, many somatic hypermutation could be introduced during affinity maturation, which cause several issues including safety and efficacy and limit their application in clinic. Here, we propose a special class of human mAbs with limited level of somatic mutations, referred to as germline-like mAbs. Remarkably, germline-like mAbs could have high affinity and potent neutralizing activity in vitro and in various animal models, despite lacking of extensive affinity maturation. Furthermore, the germline nature of these mAbs implies that they exhibit lower immunogenicity and can be elicited relatively fast in vivo compared with highly somatically mutated antibodies. In this review, we summarize germline-like mAbs with strong therapeutic and protection activity against various viruses that caused large-scale outbreaks in the last decade, including influenza virus H7N9, Zika virus (ZIKV), Dengue virus (DENV), Middle East respiratory syndrome coronavirus (MERS-CoV), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We also illustrate underlying molecular mechanisms of these germline-like antibodies against viral infections from the structural and genetic perspective, thus providing insight into further development as therapeutic agents for treatment of infectious diseases and implication for rational design of effective vaccines.

scholarly journals Computational-approach understanding the structure-function prophecy of Fibrinolytic Protease RFEA1 from Bacillus cereus RSA1

PeerJ ◽  
2021 ◽  
Vol 9 ◽  
pp. e11570
Author(s):  
Chhavi Sharma ◽  
Arti Nigam ◽  
Rajni Singh
Keyword(s):  
Structure Function ◽  
Bacillus Cereus ◽  
Structural Model ◽  
Three Dimensional ◽  
Conformational Space ◽  
Dimensional Structure ◽  
After Effects ◽  
Short Life Span ◽  
Therapeutic Enzymes

Microbial fibrinolytic proteases are therapeutic enzymes responsible to ameliorate thrombosis, a fatal cardiac-disorder which effectuates due to excessive fibrin accumulation in blood vessels. Inadequacies such as low fibrin specificity, lethal after-effects and short life-span of available fibrinolytic enzymes stimulates an intensive hunt for novel, efficient and safe substitutes. Therefore, we herewith suggest a novel and potent fibrinolytic enzyme RFEA1 from Bacillus cereus RSA1 (MK288105). Although, attributes such as in-vitro purification, characterization and thrombolytic potential of RFEA1 were successfully accomplished in our previous study. However, it is known that structure-function traits and mode of action significantly aid to commercialization of an enzyme. Also, predicting structural model of a protein from its amino acid sequence is challenging in computational biology owing to intricacy of energy functions and inspection of vast conformational space. Our present study thus reports In-silico structural-functional analysis of RFEA1. Sequence based modelling approaches such as—Iterative threading ASSEmbly Refinement (I-TASSER), SWISS-MODEL, RaptorX and Protein Homology/analogY Recognition Engine V 2.0 (Phyre2) were employed to model three-dimensional structure of RFEA1 and the modelled RFEA1 was validated by structural analysis and verification server (SAVES v6.0). The modelled crystal structure revealed the presence of high affinity Ca1 binding site, associated with hydrogen bonds at Asp147, Leu181, Ile185 and Val187residues. RFEA1 is structurally analogous to Subtilisin E from Bacillus subtilis 168. Molecular docking analysis using PATCH DOCK and FIRE DOCK servers was performed to understand the interaction of RFEA1 with substrate fibrin. Strong RFEA1-fibrin interaction was observed with high binding affinity (−21.36 kcal/mol), indicating significant fibrinolytic activity and specificity of enzyme RFEA1. Overall, the computational research suggests that RFEA1 is a subtilisin-like serine endopeptidase with proteolytic potential, involved in thrombus hydrolysis.

scholarly journals 3D-PP: A Tool for Discovering Conserved Three-Dimensional Protein Patterns

2019 ◽  
Vol 20 (13) ◽  
pp. 3174
Author(s):  
Alejandro Valdés-Jiménez ◽  
Josep-L. Larriba-Pey ◽  
Gabriel Núñez-Vivanco ◽  
Miguel Reyes-Parada
Keyword(s):  
Homology Modeling ◽  
In Silico ◽  
Rational Design ◽  
Protein Structures ◽  
Three Dimensional ◽  
Web Server ◽  
Free Access ◽  
Protein Patterns ◽  
Structural Motifs ◽  
The Web

Discovering conserved three-dimensional (3D) patterns among protein structures may provide valuable insights into protein classification, functional annotations or the rational design of multi-target drugs. Thus, several computational tools have been developed to discover and compare protein 3D-patterns. However, most of them only consider previously known 3D-patterns such as orthosteric binding sites or structural motifs. This fact makes necessary the development of new methods for the identification of all possible 3D-patterns that exist in protein structures (allosteric sites, enzyme-cofactor interaction motifs, among others). In this work, we present 3D-PP, a new free access web server for the discovery and recognition all similar 3D amino acid patterns among a set of proteins structures (independent of their sequence similarity). This new tool does not require any previous structural knowledge about ligands, and all data are organized in a high-performance graph database. The input can be a text file with the PDB access codes or a zip file of PDB coordinates regardless of the origin of the structural data: X-ray crystallographic experiments or in silico homology modeling. The results are presented as lists of sequence patterns that can be further analyzed within the web page. We tested the accuracy and suitability of 3D-PP using two sets of proteins coming from the Protein Data Bank: (a) Zinc finger containing and (b) Serotonin target proteins. We also evaluated its usefulness for the discovering of new 3D-patterns, using a set of protein structures coming from in silico homology modeling methodologies, all of which are overexpressed in different types of cancer. Results indicate that 3D-PP is a reliable, flexible and friendly-user tool to identify conserved structural motifs, which could be relevant to improve the knowledge about protein function or classification. The web server can be freely utilized at https://appsbio.utalca.cl/3d-pp/.

scholarly journals A Structural Model of Pestivirus Erns Based on Disulfide Bond Connectivity and Homology Modeling Reveals an Extremely Rare Vicinal Disulfide

2002 ◽  
Vol 76 (20) ◽  
pp. 10383-10392 ◽  
Author(s):  
J. P. M. Langedijk ◽  
P. A. van Veelen ◽  
W. M. M. Schaaper ◽  
A. H. de Ru ◽  
R. H. Meloen ◽  
...  
Keyword(s):  
Homology Modeling ◽  
Structural Model ◽  
Proteolytic Enzymes ◽  
Three Dimensional ◽  
Surface Protein ◽  
Disulfide Bridge ◽  
Ionization Mass ◽  
Three Dimensional Model ◽  
Protein Fragments ◽  
Infected Cells

ABSTRACT Erns is a pestivirus envelope glycoprotein and is the only known viral surface protein with RNase activity. Erns is a disulfide-linked homodimer of 100 kDa; it is found on the surface of pestivirus-infected cells and is secreted into the medium. In this study, the disulfide arrangement of the nine cysteines present in the mature dimer was established by analysis of the proteolytically cleaved protein. Fragments were obtained after digestion with multiple proteolytic enzymes and subsequently analyzed by liquid chromatography-electrospray ionization mass spectrometry. The analysis demonstrates which cysteine is involved in dimerization and reveals an extremely rare vicinal disulfide bridge of unknown function. With the assistance of the disulfide arrangement, a three-dimensional model was built by homology modeling based on the alignment with members of the Rh/T2/S RNase family. Compared to these other RNase family members, Erns shows an N-terminal truncation, a large insertion of a cystine-rich region, and a C-terminal extension responsible for membrane translocation. The homology to mammalian RNase 6 supports a possible role of Erns in B-cell depletion.

scholarly journals Covering complete proteomes with X-ray structures: a current snapshot

2014 ◽  
Vol 70 (11) ◽  
pp. 2781-2793 ◽  
Author(s):  
Marcin J. Mizianty ◽  
Xiao Fan ◽  
Jing Yan ◽  
Eric Chalmers ◽  
Christopher Woloschuk ◽  
...  
Keyword(s):  
Homology Modeling ◽  
Structure Determination ◽  
Large Scale ◽  
Structural Model ◽  
Target Selection ◽  
Three Dimensional ◽  
X Ray ◽  
X Ray Crystallography ◽  
Knowledge Based ◽  
Wide Range

Structural genomics programs have developed and applied structure-determination pipelines to a wide range of protein targets, facilitating the visualization of macromolecular interactions and the understanding of their molecular and biochemical functions. The fundamental question of whether three-dimensional structures of all proteins and all functional annotations can be determined using X-ray crystallography is investigated. A first-of-its-kind large-scale analysis of crystallization propensity for all proteins encoded in 1953 fully sequenced genomes was performed. It is shown that current X-ray crystallographic knowhow combined with homology modeling can provide structures for 25% of modeling families (protein clusters for which structural models can be obtained through homology modeling), with at least one structural model produced for each Gene Ontology functional annotation. The coverage varies between superkingdoms, with 19% for eukaryotes, 35% for bacteria and 49% for archaea, and with those of viruses following the coverage values of their hosts. It is shown that the crystallization propensities of proteomes from the taxonomic superkingdoms are distinct. The use of knowledge-based target selection is shown to substantially increase the ability to produce X-ray structures. It is demonstrated that the human proteome has one of the highest attainable coverage values among eukaryotes, and GPCR membrane proteins suitable for X-ray structure determination were determined.

scholarly journals Biochemical Analysis of Phytolacca DOPA Dioxygenase

2015 ◽  
Vol 10 (5) ◽  
pp. 1934578X1501000
Author(s):  
Kana Takahashi ◽  
Kazuko Yoshida ◽  
Kei Yura ◽  
Hiroshi Ashihara ◽  
Masaaki Sakuta
Keyword(s):  
Biochemical Analysis ◽  
Structural Model ◽  
Specific Activity ◽  
Three Dimensional ◽  
Single Amino Acid ◽  
Phytolacca Americana ◽  
Conserved Motif ◽  
Single Amino Acid Residue ◽  
The Difference

The biochemical analysis of Phytolacca americana DOPA dioxygenases (PaDOD1 and PaDOD2) was carried out. The recombinant protein of PaDOD1 catalyzed the conversion of DOPA to betalamic acid, whereas DOD activity was not detected in PaDOD2 in vitro. While the reported motif conserved in DODs from betalain-producing plants was found in PaDOD1, a single amino acid residue alteration was detected in PaDOD2. A mutated PaDOD1 protein with a change of 177 Asn to Gly showed reduced specific activity compared with PaDOD1, while DOPA dioxygenase activity was not observed for a mutated PaDOD2 protein which had its conserved motif replaced with that of PaDOD1. A three-dimensional (3D) structural model of PaDOD1 and PaDOD2 showed that the conserved motif in DODs was located in the N-terminal side of a loop, which was found close to the putative active site. The difference in stability of the loop may affect the enzymatic activity of PaDOD2.

scholarly journals HOMOLOGY MODELING OF SUBCUTANEOUS FILARIASIS DHFR PROTEINS

2017 ◽  
Vol 9 (6) ◽  
pp. 76
Author(s):  
Kavita Chandramore
Keyword(s):  
Homology Modeling ◽  
Protein Function ◽  
Structural Model ◽  
Query Sequence ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Parasitic Disease ◽  
Loa Loa ◽  
Knowledge Based ◽  
Template Sequence

Objective: A systematic technique for protein modeling offers great assistance in the study of protein function, dynamics, interactions with ligands, other proteins and even in drug discovery and drug design. Subcutaneous filariasis is rare parasitic disease caused by Loa Loa (eye worm) and monosonallastreptoscerca species. Methods: The present study develop three dimensional structure of dihydrofolatereductase present in Loa loa species. For this purpose knowledge based homology modeling is used by using Schrodinger Glide 5.6 software.Results: The procedure involves alignment that maps residues in the query sequence to residues in the template sequence to generate structural model of target, which was further refined and final result validated by using Ramchandran plot.Conclusion: In ramchandran plot majority of the amino acids are in the phi-psi distribution and thedevelop model is reliable and of good quality.

scholarly journals Rational Design of a Bifunctional AND-Gate Ligand to Modulate Cell-Cell Interactions

2019 ◽  
Author(s):  
Jungmin Lee ◽  
Andyna Vernet ◽  
Katherine Redfield ◽  
Shulin Lu ◽  
Ionita C. Ghiran ◽  
...  
Keyword(s):  
Rational Design ◽  
Structural Model ◽  
Molecular Geometry ◽  
Glycophorin A ◽  
Cell Type ◽  
V Region ◽  
Meso Scale ◽  
Signaling Activity

ABSTRACTProtein “AND-gate” systems, in which a ligand acts only on cells with two different receptors, direct signaling activity to a particular cell type and avoid action on other cells. In a bifunctional AND-Gate protein, the molecular geometry of the protein domains is crucial. Here we constructed a tissue-targeted erythropoietin (EPO) that stimulates red blood cell (RBC) production without triggering thrombosis. EPO was directed to RBC precursors and mature RBCs by fusion to an anti-glycophorin A antibody V region. Many such constructs activated EPO receptorsin vitroand stimulated RBC and not platelet production in mice but nonetheless enhanced thrombosis in mice and caused adhesion between RBCs and EPO receptor-bearing cells. Based on a protein-structural model of the RBC surface, we rationally designed an anti-glycophorin/EPO fusion that does not induce cell adhesionin vitroor enhance thrombosisin vivo. Thus, meso-scale geometry can inform design of synthetic-biological systems.

scholarly journals The sclerostin-neutralizing antibody AbD09097 recognizes an epitope adjacent to sclerostin's binding site for the Wnt co-receptor LRP6

Open Biology ◽  
2016 ◽  
Vol 6 (8) ◽  
pp. 160120 ◽  
Author(s):  
V. Boschert ◽  
C. Frisch ◽  
J. W. Back ◽  
K. van Pee ◽  
S. E. Weidauer ◽  
...  
Keyword(s):  
Rational Design ◽  
Bone Growth ◽  
Interaction Analysis ◽  
Neutralizing Antibody ◽  
Affinity Maturation ◽  
Negative Regulator ◽  
Binding Motif ◽  
Biochemical Analyses ◽  
Set Up

The glycoprotein sclerostin has been identified as a negative regulator of bone growth. It exerts its function by interacting with the Wnt co-receptor LRP5/6, blocks the binding of Wnt factors and thereby inhibits Wnt signalling. Neutralizing anti-sclerostin antibodies are able to restore Wnt activity and enhance bone growth thereby presenting a new osteoanabolic therapy approach for diseases such as osteoporosis. We have generated various Fab antibodies against human and murine sclerostin using a phage display set-up. Biochemical analyses have identified one Fab developed against murine sclerostin, AbD09097 that efficiently neutralizes sclerostin's Wnt inhibitory activity. In vitro interaction analysis using sclerostin variants revealed that this neutralizing Fab binds to sclerostin's flexible second loop, which has been shown to harbour the LRP5/6 binding motif. Affinity maturation was then applied to AbD09097, providing a set of improved neutralizing Fab antibodies which particularly bind human sclerostin with enhanced affinity. Determining the crystal structure of AbD09097 provides first insights into how this antibody might recognize and neutralize sclerostin. Together with the structure–function relationship derived from affinity maturation these new data will foster the rational design of new and highly efficient anti-sclerostin antibodies for the therapy of bone loss diseases such as osteoporosis.

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