Biology subjects

Shafiei, A.

Publications and source records attributed to Shafiei, A..

8 recordsLinked to original sources

bioRxiv2025

The N-terminal order-disorder transition is a critical determinant for a metamorphosis of IscU

IscU, a key scaffold protein mediating the biogenesis of iron-sulfur (Fe-S) clusters, exhibits metamorphic characteristics crucial for its versatile and efficient function. Previous studies have demonstrated that IscU has two interconverting conformations: the structured state (S-state) and the disordered state (D-state), each contributing to its distinct functionality and interaction network. Despite its physiological importance, the precise mechanism underpinning the maintenance of IscUs unique structural heterogeneity has remained elusive. In this study, we used computational and experimental approaches to reveal that the N-terminal order-disorder plays a critical role in the metamorphic modulation of IscU. We found that the N-terminal region displays greater structural plasticity, which is linked to other regions of IscU through coevolutionary relationships. Moreover, we demonstrated that the degree of orderliness in the N-terminal region correlates positively with the stabilization of IscUs S-state and negatively with its affinity for HscA. This indicates that the flexibility in the N-terminal region is finely tuned to optimize IscUs physiological efficiency and efficacy. Finally, our data suggest that a peptide mimicking the N-terminal motif can modulate IscUs metamorphic properties, suggesting a novel therapeutic potential for related pathogenic processes.

biochemistry↗

bioRxiv2024

Genomic alterations and transcriptional phenotypes in circulating tumor DNA and matched metastatic tumor

BackgroundProfiling circulating cell-free DNA (cfDNA) has become a fundamental practice in cancer medicine, but the effectiveness of cfDNA at elucidating tumor-derived molecular features has not been systematically compared to standard single-lesion tumor biopsies in prospective cohorts of patients. The use of plasma instead of tissue to guide therapy is particularly attractive for patients with small cell lung cancer (SCLC), a cancer whose aggressive clinical course making it exceedingly challenging to obtain tumor biopsies. MethodsHere, a prospective cohort of 49 plasma samples obtained before, during, and after treatment from 20 patients with recurrent SCLC, we study cfDNA low pass whole genome (0.1X coverage) and exome (130X) sequencing in comparison with time-point matched tumor, characterized using exome and transcriptome sequencing. ResultsDirect comparison of cfDNA versus tumor biopsy reveals that cfDNA not only mirrors the mutation and copy number landscape of the corresponding tumor but also identifies clinically relevant resistance mechanisms and cancer driver alterations not found in matched tumor biopsies. Longitudinal cfDNA analysis reliably tracks tumor response, progression, and clonal evolution. Genomic sequencing coverage of plasma DNA fragments around transcription start sites shows distinct treatment-related changes and captures the expression of key transcription factors such as NEUROD1 and REST in the corresponding SCLC tumors, allowing prediction of SCLC neuroendocrine phenotypes and treatment responses. Conclusions These findings have important implications for non-invasive stratification and subtype-specific therapies for patients with SCLC, now treated as a single disease.

cancer biology↗

bioRxiv2023

Ambient Temperature Bacterial Large Ribosomal Subunit Structure Enabled by Serial Femtosecond X-ray Crystallography

Ribosomes are the supramolecular complexes responsible for protein synthesis. The large 50S ribosomal subunit catalyzes the peptidyl transferase reaction and peptide bond formation between amino acids. The 50S is targeted by many known clinically effective antibiotics. Available structures, obtained at cryogenic temperatures (CT), are used for drug discovery despite that active or important target sites may display a structural configuration that is CT-induced. The introduction of ultrafast and ultrabright X-ray free electron laser (XFEL) pulses has enabled the structural observation of biological macro- and supramolecules at previously unattainable, near-physiological temperatures. In this study, we use ultrafast and ultrabright XFEL pulses to solve the apo form of 50S ribosomal subunit isolated from the extremely thermophilic bacterium Thermus thermophilus at ambient temperature (AT). The dimeric structure of the 50S subunit presented in this work is among the largest ([~]3 megadalton) structures determined using an XFEL source to date. This study demonstrates the ability to obtain new information about ribosome structural dynamics at AT through serial femtosecond X-ray crystallography (SFX). This allowed us to capture previously unobserved dynamics of ribosomal protein uL23 and coordination by hexahydrated magnesium cations at a hitherto unseen resolution at near-physiological temperature. Also, residue A2602, at the core of the peptidyl transferase center (PTC), shows a rather different orientation of the sugar moiety if compared to CT structures. In addition, our structure highlights the importance of flexible residues at both the PTC and in the binding sites for antibiotics erythromycin and chloramphenicol. The method implemented here may also serve as a starting point for future structural research involving the 50S subunit complexes by employing time-resolved mix-inject and probe kineto-crystallography experiments at XFELs. Unveiling ligand-dependent 50S dynamics at physiological temperatures shall guide further development of next-generation antibiotics that target the translation machinery.

biophysics↗

bioRxiv2023

Ambient temperature crystal structure of Escherichia coli CyaY protein displays alternate conformation

Frataxin is a 23 KDa mitochondrial iron-binding protein that is involved in biogenesis of iron sulfur clusters. A deficiency in frataxin leads to Friedreichs ataxia, a progressive neurodegenerative disorder. The bacterial ortholog of eukaryotic mitochondrial frataxin, CyaY, is thought to play a role in iron sulfur cluster assembly as an iron supplier, making it an important target for study. Here, we present the first ambient temperature crystal structure of CyaY protein from Escherichia coli, obtained using the Turkish Light Source "Turkish DeLight". This study reveals the dynamic structural characteristics of CyaY at near-physiological temperature and displays an alternate conformation, highlighting the importance of temperature considerations in protein structure characterization and providing new insights into the proteins flexibility.

biochemistry↗

bioRxiv2022

Rapid and High Resolution Ambient Temperature Structure Determination at Turkish Light Source

High-resolution biomacromolecular structure determination is essential to better understand protein function and dynamics. Serial crystallography is an emerging structural biology technique which has fundamental limitations due to either sample volume requirements or immediate access to the competitive X-ray beamtime. Obtaining a high volume of well-diffracting, sufficient-size crystals while mitigating radiation damage remains a critical bottleneck of serial crystallography. As an alternative, we introduce the plate-reader module adapted for using a 72-well Terasaki plate for biomacromolecule structure determination at a convenience of a home X-ray source. We also present the first ambient temperature lysozyme structure determined at the Turkish Light Source (Turkish DeLight). The complete dataset was collected in 18.5 mins with resolution extending to 2.39 [A] and 100% completeness. Combined with our previous cryogenic structure (PDB ID: 7Y6A), the ambient temperature structure provides invaluable information about the structural dynamics of the lysozyme. Turkish DeLight provides robust and rapid ambient temperature biomacromolecular structure determination with limited radiation damage.

biophysics↗

bioRxiv2022

Cryogenic X-ray crystallographic studies of biomacromolecules at Turkish Light Source: Turkish DeLight

X-ray crystallography is a robust and powerful structural biology technique that provides high-resolution atomic structures of biomacromolecules. Scientists use this technique to unravel mechanistic and structural details of biological macromolecules (e.g. proteins, nucleic acids, protein complexes, protein-nucleic acid complexes, or large biological compartments). Since its inception, single-crystal cryo-crystallography has never been performed in Turkiye due to the lack of a single-crystal X-ray diffractometer. The X-ray diffraction facility recently established at the University of Health Sciences, Istanbul, Turkiye will enable Turkish and international researchers to easily perform high-resolution structural analysis of biomacromolecules from single crystals. Here, we describe the technical and practical outlook of a state-of-the-art home-source X-ray, using lysozyme as a model protein. The methods and practice described in this article can be applied to any biological sample for structural studies. Therefore, this article will be a valuable practical guide from sample preparation to data analysis.

biophysics↗

bioRxiv2021

Structural insight into host plasma membrane association and assembly of HIV-1 Matrix protein

HIV-1 continues to be a global health concern since AIDS was first recognized by the World Health Organization (WHO). It is estimated that there were 38 million people infected with HIV-1 and 1.5 million deaths in 2019 alone. A better understanding of the details of the HIV late-stage life cycle, involving Pr55Gag attachment to the membrane for the further oligomerization to release virion, will provide us new avenues for potential treatment. Inositol hexakisphosphate (IP6) is an abundant endogenous cyclitol molecule and its binding was linked to the oligomerization of Pr55Gag via the MA domain. However, the binding site of IP6 on MA was unknown and the structural details of this interaction were missing. Here, we present three high-resolution crystal structures of the MA domain in complex with IP6 molecules to reveal its binding mode. Additionally, extensive Differential Scanning Fluorimetry analysis combined with cryo- and ambient-temperature X-ray crystallography and computational biology identify the key residues that participate in IP6 binding. Our data provide novel insights about the multilayered HIV-1 virion assembly process that involves the interplay of IP6 with PIP2, a phosphoinositide essential for the membrane binding of Pr55Gag. IP6 and PIP2 have neighboring alternate binding sites within the same highly basic region (residues 18-33). This indicates that IP6 and PIP2 bindings are not mutually exclusive and may play a key role in coordinating virion particles membrane localization. Based on our three different IP6-MA complex crystal structures, we propose a new model that involves the IP6 coordination of the oligomerization of outer MA and inner CA domain 2D layers during assembly and budding.

microbiology↗

bioRxiv2020

Near-Physiological-Temperature Serial Femtosecond X-ray Crystallography Reveals Novel Conformations of SARS-CoV-2 Main Protease Active Site for Improved Drug Repurposing

The COVID19 pandemic has resulted in 25+ million reported infections and nearly 850.000 deaths. Research to identify effective therapies for COVID19 includes: i) designing a vaccine as future protection; ii) structure-based drug design; and iii) identifying existing drugs to repurpose them as effective and immediate treatments. To assist in drug repurposing and design, we determined two apo structures of Severe Acute Respiratory Syndrome CoronaVirus-2 main protease at ambienttemperature by Serial Femtosecond X-ray crystallography. We employed detailed molecular simulations of selected known main protease inhibitors with the structures and compared binding modes and energies. The combined structural biology and molecular modeling studies not only reveal the dynamics of small molecules targeting main protease but will also provide invaluable opportunities for drug repurposing and structure-based drug design studies against SARS-CoV-2. One Sentence SummaryRadiation-damage-free high-resolution SARS-CoV-2 main protease SFX structures obtained at near-physiological-temperature offer invaluable information for immediate drug-repurposing studies for the treatment of COVID19.

biophysics↗