Biology subjects

DeMirci, H.

Publications and source records attributed to DeMirci, H..

24 records · Page 2Linked to original sources

bioRxiv2021

Case Study of High-Throughput Drug Screening and Remote Data Collection for SARS-CoV-2 Main Protease by Using Serial Femtosecond X-ray Crystallography

Since early 2020, COVID-19 has grown to affect the lives of billions globally. A worldwide investigation has been ongoing for characterizing the virus and also for finding an effective drug and developing vaccines. As time has been of the essence, a crucial part of this research has been drug repurposing; therefore, confirmation of in-silico drug screening studies has been carried out for this purpose. Here we demonstrated the possibility of screening a variety of drugs efficiently by leveraging a high data collection rate of 120 images/second with the new low-noise, high dynamic range ePix10k2M Pixel Array Detector installed at the Macromolecular Femtosecond Crystallography (MFX) instrument at the Linac Coherent Light Source (LCLS). The X-ray Free-Electron Laser (XFEL) is used for remote high-throughput data collection for drug repurposing of the main protease (Mpro) of SARS-CoV-2 at ambient temperature with mitigated X-ray radiation damage. We obtained multiple structures soaked with 9 drug candidate molecules in two crystal forms. Although our drug binding attempts failed, we successfully established a high-throughput Serial Femtosecond X-ray crystallographic (SFX) data collection protocol.

molecular biology↗

bioRxiv2021

Cooperative Allostery and Structural Dynamics of Streptavidin at Cryogenic- and Ambient-temperature

Multimeric protein assemblies are abundant in nature. Streptavidin is an attractive protein that provides a paradigm system to investigate the intra- and intermolecular interactions of multimeric protein complexes. Also, it offers a versatile tool for biotechnological applications. Here, we present two apo-streptavidin structures, the first one is an ambient temperature Serial Femtosecond X-ray crystal (Apo-SFX) structure at 1.7 [A] resolution and the second one is a cryogenic crystal structure (Apo-Cryo) at 1.1 [A] resolution. These structures are mostly in agreement with previous structural data. Combined with computational analysis, these structures provide invaluable information about structural dynamics of apo streptavidin. Collectively, these data further reveal a novel cooperative allostery of streptavidin which binds to substrate via water molecules that provide a polar interaction network and mimics the substrate biotin which displays one of the strongest affinities found in nature.

biophysics↗

bioRxiv2021

Structural Insights into Bifunctional Thaumarchaeal Crotonyl-CoA Hydratase and 3-Hydroxypropionyl-CoA Dehydratase from Nitrosopumilus maritimus

The ammonia-oxidizing thaumarchaeal 3-hydroxypropionate/4-hydroxybutyrate (3HP/4HB) cycle is one of the most energy-efficient CO2 fixation cycles discovered thus far. The protein encoded by Nmar_1308 (from Nitrosopumilus maritimus SCM1) is a promiscuous enzyme that catalyzes two essential reactions within the thaumarchaeal 3HP/4HB cycle, functioning as both a crotonyl-CoA hydratase (CCAH) and 3- hydroxypropionyl-CoA dehydratase (3HPD). In performing both hydratase and dehydratase activities, Nmar_1308 reduces the total number of enzymes necessary for CO2 fixation in Thaumarchaeota, reducing the overall cost for biosynthesis. Here, we present the first high-resolution crystal structure of this bifunctional enzyme with key catalytic residues in the thaumarchaeal 3HP/4HB pathway.

biochemistry↗

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

Structural adaptation of oxygen tolerance in 4-hydroxybutyrl-CoA dehydratase, a key enzyme of archaeal carbon fixation

Autotrophic microorganisms that convert inorganic carbon into organic matter were key players in the evolution of life on Earth. As the early atmosphere became oxygenated, microorganisms needed to develop mechanisms for oxygen protection, especially those relying on enzymes containing oxygen-sensitive metal clusters (e.g., Fe-S). Here we investigated how 4-hydroxybutyryl-CoA dehydratase (4HBD) - the key enzyme of the 3-hydroxypropionate/4-hydroxybutyrate (HP/HB) cycle for CO2-fixation - adapted as conditions shifted from anoxic to oxic. 4HBD is found in both anaerobic bacteria and aerobic ammonia-oxidizing archaea (AOA). The oxygen-sensitive bacterial 4HBD and oxygen-tolerant archaeal 4HBD share 59 % amino acid identity. To examine the structural basis of oxygen tolerance in archaeal 4HBD, we determined the atomic resolution structure of the enzyme. Two tunnels providing access to the canonical [4Fe-4S] cluster in oxygen-sensitive bacterial 4HBD were closed with four conserved mutations found in all aerobic AOA and other archaea. Further biochemical experiments and molecular dynamics simulations support our findings that restricting access to the active site is the key to oxygen tolerance, explaining how active site evolution drove a major evolutionary transition. Significance statementAutotrophy (primary production) was the first life strategy on Earth. Before photosynthesis (using solar energy to fix carbon dioxide), life relied on chemical reactions for energy. These chemosynthetic reactions are present in all domains of life, including archaea possessing the most energy-efficient carbon fixation pathway - the 3-hydroxypropionate/4-hydroxybutyrate cycle. This efficiency results from enzyme modifications, including enhanced enzyme stability and catalysis of multiple reactions. We reveal the first structure of aerobic 4-hydroxybutyryl-CoA dehydratase (4HBD) from ammonia-oxidizing archaea. These archaea are among the most abundant organisms on the planet, and their 4HBD active site evolved oxygen tolerance to support aerobic metabolism. This modification can provide further insight into enzyme evolution on early earth, as photosynthesis developed and began oxygenating the atmosphere.

evolutionary biology↗

bioRxiv2018

Aminoglycoside ribosome interactions reveal novel conformational states at ambient temperature

The bacterial 30S ribosomal subunit is a primary antibiotic target. Despite decades of discovery, the mechanisms by which antibiotic binding induces ribosomal dysfunction are not fully understood. Ambient temperature crystallographic techniques allow more biologically relevant investigation of how local antibiotic binding site interactions trigger global subunit rearrangements that perturb protein synthesis. Here, the structural effects of 2-deoxystreptamine (paromomycin and sisomicin), a novel sisomicin derivative, N1-methyl sulfonyl sisomicin (N1MS) and the non-deoxystreptamine (streptomycin) aminoglycosides on the ribosome at ambient and cryogenic temperatures were examined. Comparative studies led to three main observations. First, individual aminoglycoside-ribosome interactions in the decoding center were similar for cryogenic vs ambient temperature structures. Second, analysis of a highly conserved GGAA tetraloop of h45 revealed aminoglycoside-specific conformational changes, which are affected by temperature only for N1MS. We report the h44/h45 interface in varying states, that is, engaged, disengaged and in equilibrium. Thirdly, we observe aminoglycoside-induced effects on 30S domain closure, including a novel intermediary closure state, which is also sensitive to temperature. Analysis of three ambient and five cryogenic crystallography datasets reveal a correlation between h44/h45 engagement and domain closure. These observations illustrate the role of ambient temperature crystallography in identifying dynamic mechanisms of ribosomal dysfunction induced by local drug-binding site interactions. Together these data identify tertiary ribosomal structural changes induced by aminoglycoside binding that provides functional insight and targets for drug design.

biophysics↗