Biology subjects

Sierra, R. G.

Publications and source records attributed to Sierra, R. G..

At least 19 recordsLinked to original sources

bioRxiv2026

Conformational flexibility of soybean lipoxygenase is coupled to crystal solvent content in serial crystallography

X-ray crystallography is increasingly employed to study protein conformational ensembles under physiological conditions, but the effects of the crystal lattice on protein motions remains understudied. Here, we report the structure determination of soybean lipoxygenase-1 (SLO) from microcrystal slurries using serial femtosecond crystallography (SFX) at the Linac Coherent Light Source. During data analysis, we observed unexpected polymorphism in SLOs unit-cell parameters, arising from two compounding factors: indexing ambiguities caused by the pseudo-tetragonal symmetry of the SLO crystal lattice, and true non-isomorphism between individual crystal populations consistent with different solvent content. By combining unit-cell clustering with systematic reindexing, we resolved two distinct polymorphs and determined two independent structures from a single experiment. The two structures exhibit a small overall RMSD (0.34 [A]), yet a difference distance matrix reveals coordinated rearrangements that are not readily apparent from simple structural overlays. Furthermore, a difference of approximately 8.5% in crystal solvent content produces measurable differences in crystal contacts and conformational flexibility. The more hydrated (large-cell) polymorph exhibits greater inter-domain flexibility, as well as higher B-factors in key hydrophobic core residues. In the dehydrated (small-cell) polymorph, which shows less interdomain flexibility, these same residues adopt discrete alternative conformations resolvable in the electron density. Our results highlight that subtle changes in crystal packing can give rise to distinct conformational landscapes for crystallized proteins, with potential implications for the interpretation of protein intramolecular dynamics from crystallographic data. SynopsisUsing soybean lipoxygenase-1 as a model system, we determine two independent structures within a single experiment that differ in crystal solvent content. Despite a small overall RMSD, a detailed analysis reveals coordinated domain-level rearrangements and shows that conformational flexibility is sensitive to crystal packing, a consideration of broad relevance for the study of protein dynamics using X-ray crystallography.

biochemistry↗

bioRxiv2023

4D Crystallography Captures Transient IF1-Ribosome Dynamics in Translation Initiation

Initiation factor 1 (IF1) is one of multiple key ligands involved in the initiation of mRNA translation, a highly dynamic and carefully-orchestrated process. However, details surrounding IF1 transient interactions with the small 30S ribosomal subunit remain incompletely understood despite characterization of unbound and fully-bound 30S states. Improvements in X-ray light sources and crystallographic techniques are now enabling time-resolved structural studies at near-physiological temperature and near-atomic resolution and thus the structural investigation of such dynamic processes. Here, we employed time-resolved serial femtosecond X-ray crystallography (TR-SFX) to probe the binding of IF1 to the small 30S ribosomal subunit in real time. Our time-resolved structural data demonstrates transient cryptic short-, mid-, and long-range allostery among different regions of the small 30 ribosomal subunit during IF1 binding, revealing small- and large-scale protein-target interactions and dynamics within intermediate macromolecular states at unprecedented temporal and spatial resolution. These data represent one of the first such 4D crystallographic studies assessing protein-protein and protein-RNA interactions and could serve as the basis for subsequent studies of the ribosome and of the multitudinous dynamic processes which underpin biology, and therefore, of life.

biophysics↗

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

Changes in an Enzyme Ensemble During Catalysis Observed by High Resolution XFEL Crystallography

Enzymes populate ensembles of structures with intrinsically different catalytic proficiencies that are difficult to experimentally characterize. We use time-resolved mix-and-inject serial crystallography (MISC) at an X-ray free electron laser (XFEL) to observe catalysis in a designed mutant (G150T) isocyanide hydratase (ICH) enzyme that enhances sampling of important minor conformations. The active site exists in a mixture of conformations and formation of the thioimidate catalytic intermediate selects for catalytically competent substates. A prior proposal for active site cysteine charge-coupled conformational changes in ICH is validated by determining structures of the enzyme over a range of pH values. A combination of large molecular dynamics simulations of the enzyme in crystallo and time-resolved electron density maps shows that ionization of the general acid Asp17 during catalysis causes additional conformational changes that propagate across the dimer interface, connecting the two active sites. These ionization-linked changes in the ICH conformational ensemble permit water to enter the active site in a location that is poised for intermediate hydrolysis. ICH exhibits a tight coupling between ionization of active site residues and catalysis-activated protein motions, exemplifying a mechanism of electrostatic control of enzyme dynamics.

biochemistry↗

bioRxiv2023

Detection of a geminate photoproduct of bovine cytochrome c oxidase by time-resolved serial femtosecond crystallography

Cytochrome c oxidase (CcO) is a large membrane-bound hemeprotein that catalyzes the reduction of dioxygen to water. Unlike classical dioxygen binding hemeproteins with a heme b group in their active sites, CcO has a unique binuclear center (BNC) comprised of a copper atom (CuB) and a heme a3 iron, where O2 binds and is reduced to water. CO is a versatile O2 surrogate in ligand binding and escape reactions. Previous time-resolved spectroscopic studies of the CO complexes of bovine CcO (bCcO) revealed that photolyzing CO from the heme a3 iron leads to a metastable intermediate (CuB-CO), where CO is bound to CuB, before it escapes out of the BNC. Here, with a time-resolved serial femtosecond X-ray crystallography-based pump-probe method, we detected a geminate photoproduct of the bCcO-CO complex, where CO is dissociated from the heme a3 iron and moved to a temporary binding site midway between the CuB and the heme a3 iron, while the locations of the two metal centers and the conformation of the Helix-X, housing the proximal histidine ligand of the heme a3 iron, remain in the CO complex state. This new structure, combined with other reported structures of bCcO, allows the full definition of the ligand dissociation trajectory, as well as the associated protein dynamics.

biophysics↗

bioRxiv2023

Structural basis for functional properties of cytochrome c oxidase

Cytochrome c oxidase (CcO) is an essential enzyme in mitochondrial and bacterial respiration. It catalyzes the four-electron reduction of molecular oxygen to water and harnesses the chemical energy to translocate four protons across biological membranes, thereby establishing the proton gradient required for ATP synthesis1. The full turnover of the CcO reaction involves an oxidative phase, in which the reduced enzyme (R) is oxidized by molecular oxygen to the metastable oxidized OH state, and a reductive phase, in which OH is reduced back to the R state. During each of the two phases, two protons are translocated across the membranes2. However, if OH is allowed to relax to the resting oxidized state (O), a redox equivalent to OH, its subsequent reduction to R is incapable of driving proton translocation2,3. How the O state structurally differs from OH remains an enigma in modern bioenergetics. Here, with resonance Raman spectroscopy and serial femtosecond X-ray crystallography (SFX)4, we show that the heme a3 iron and CuB in the active site of the O state, like those in the OH state5,6, are coordinated by a hydroxide ion and a water molecule, respectively. However, Y244, a residue covalently linked to one of the three CuB ligands and critical for the oxygen reduction chemistry, is in the neutral protonated form, which distinguishes O from OH, where Y244 is in the deprotonated tyrosinate form. These structural characteristics of O provide new insights into the proton translocation mechanism of CcO.

biophysics↗

bioRxiv2022

Heterogeneity in the M. tuberculosis β-lactamase inhibition by Sulbactam

For decades, researchers have been determined to elucidate essential enzymatic functions on the atomic lengths scale by tracing atomic positions in real time. Our work builds on new possibilities unleashed by mix-and-inject serial crystallography (MISC)1-5 at X-ray free electron laser facilities. In this approach, enzymatic reactions are triggered by mixing substrate or ligand solutions with enzyme microcrystals6. Here, we report in atomic detail and with millisecond time-resolution how the Mycobacterium tuberculosis enzyme BlaC is inhibited by sulbactam (SUB). Our results reveal ligand binding heterogeneity, ligand gating7-9, cooperativity, induced fit10,11 and conformational selection11-13 all from the same set of MISC data, detailing how SUB approaches the catalytic clefts and binds to the enzyme non-covalently before reacting to a trans-enamine. This was made possible in part by the application of the singular value decomposition14 to the MISC data using a newly developed program that remains functional even if unit cell parameters change during the reaction.

molecular biology↗

bioRxiv2022

Mapping Protein Dynamics at High-Resolution with Temperature-Jump X-ray Crystallography

Understanding and controlling protein motion at atomic resolution is a hallmark challenge for structural biologists and protein engineers because conformational dynamics are essential for complex functions such as enzyme catalysis and allosteric regulation. Time-resolved crystallography offers a window into protein motions, yet without a universal perturbation to initiate conformational changes the method has been limited in scope. Here we couple a solvent-based temperature jump with time-resolved crystallography to visualize structural motions in lysozyme, a dynamic enzyme. We observed widespread atomic vibrations on the nanosecond timescale, which evolve on the sub-millisecond timescale into localized structural fluctuations that are coupled to the active site. An orthogonal perturbation to the enzyme, inhibitor binding, altered these dynamics by blocking key motions that allow energy to dissipate from vibrations into functional movements linked to the catalytic cycle. Because temperature-jump is a universal method for perturbing molecular motion, the method demonstrated here is broadly applicable for studying protein dynamics.

biophysics↗

bioRxiv2021

De novo determination of mosquitocidal Cry11Aa and Cry11Ba structures from naturally-occurring nanocrystals

Cry11Aa and Cry11Ba are the two most potent toxins produced by mosquitocidal Bacillus thuringiensis subsp. israelensis and jegathesan, respectively. The toxins naturally crystallize within the host; however, the crystals are too small for structure determination at synchrotron sources. Therefore, we applied serial femtosecond crystallography at X-ray free electron lasers to in vivo-grown nanocrystals of these toxins. The structure of Cry11Aa was determined de novo using the single-wavelength anomalous dispersion method, which in turn enabled the determination of the Cry11Ba structure by molecular replacement. The two structures reveal a new pattern for in vivo crystallization of Cry toxins, whereby each of their three domains packs with a symmetrically identical domain, and a cleavable crystal packing motif is located within the protoxin rather than at the termini. The diversity of in vivo crystallization patterns suggests explanations for their varied levels of toxicity and rational approaches to improve these toxins for mosquito control.

molecular biology↗

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 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↗

bioRxiv2019

Comparing serial X-ray crystallography and microcrystal electron diffraction (MicroED) as methods for routine structure determination from small macromolecular crystals

Innovative new crystallographic methods are facilitating structural studies from ever smaller crystals of biological macromolecules. In particular, serial X-ray crystallography and microcrystal electron diffraction (MicroED) have emerged as useful methods for obtaining structural information from crystals on the nanometer to micron scale. Despite the utility of these methods, their implementation can often be difficult, as they present many challenges not encountered in traditional macromolecular crystallography experiments. Here, we describe XFEL serial crystallography experiments and MicroED experiments using batch-grown microcrystals of the enzyme cyclophilin A (CypA). Our results provide a roadmap for researchers hoping to design macromolecular microcrystallography experiments, and they highlight the strengths and weaknesses of the two methods. Specifically, we focus on how the different physical conditions imposed by the sample preparation and delivery methods required for each type of experiment effect the crystal structure of the enzyme.

biophysics↗

bioRxiv2019

Coupled inter-subunit dynamics enable the fastest CO2-fixation by reductive carboxylases

Enoyl-CoA carboxylases/reductases (ECRs) belong to the most efficient CO2-fixing enzymes described to date. However, the molecular mechanisms underlying ECRs extraordinary catalytic activity on the level of the protein assembly remain elusive. Here we used a combination of ambient temperature X-ray Free Electron Laser (XFEL) and cryogenic synchrotron experiments to study the structural organization of the ECR from Kitasatospora setae. K. setae ECR is a homo-tetramer that differentiates into a dimer of dimers of open- and closed-form subunits in the catalytically active state. Using molecular dynamics simulations and structure-based mutagenesis, we show that catalysis is synchronized in K. setae ECR across the pair of two dimers. This conformational coupling of catalytic domains is conferred by individual amino acids to achieve high CO2-fixation rates. Our results provide unprecedented insights into the dynamic organization and synchronized inter- and intra-subunit communications of this remarkably efficient CO2-fixing enzyme during catalysis. Significance StatementFixation of CO2 offers real potential for reaching negative CO2 emissions in bioenergy, and bioproduct utilization. The capture and conversion of atmospheric CO2 remains a challenging task. Existing biological systems can be exploited and optimized for this use. Bacterial enoyl-CoA carboxylases/reductases (ECRs) encompass the fastest CO2-fixing enzymes found in nature to date. However, the mechanisms underlying ECRs extraordinary catalytic activity remain elusive. Our structural, computational, and biochemical results elucidate the dynamic structural organization of the ECR complex and describe how coupled motions of catalytic domains in the ECR tetramer drive carboxylation. This mechanistic understanding is critical for engineering highly efficient CO2-fixing biocatalysts for bioenergy and bioproduct applications.

biophysics↗

bioRxiv2019

Serial femtosecond X-ray diffraction of HIV-1 Gag MA-IP6 microcrystals at ambient temperature

The Human immunodeficiency virus-1 (HIV-1) matrix (MA) domain is involved in the highly regulated assembly process of the virus particles that occur at the host cells plasma membrane. High-resolution structures of the MA domain determined using cryo X- ray crystallography have provided initial insights into the possible steps in the viral assembly process. However, these structural studies have relied on large and frozen crystals in order to reduce radiation damage caused by the intense X-rays. Here, we report the first XFEL study of the HIV-1 MA domains interaction with inositol hexaphosphate (IP6), a phospholipid headgroup mimic. We also describe the purification, characterization and microcrystallization of two MA crystal forms obtained in the presence of IP6. In addition, we describe the capabilities of serial femtosecond X-ray crystallography (SFX) using X-ray free-electron laser (XFEL) to elucidate the diffraction data of MA-IP6 complex microcrystals in liquid suspension at ambient temperature. Two different microcrystal forms of MA-IP6 complex both diffracted to beyond 3.5 [A] resolution, demonstrating the feasibility of using SFX to study the complexes of MA domain of HIV-1 Gag polyprotein with IP6 at near-physiological temperatures. Further optimization of the experimental and data analysis procedures will lead to better understanding of the MA domain of HIV-1 Gag and IP6 interaction at high resolution and provide basis for optimization of the lead compounds for efficient inhibition of the Gag protein recruitment to the plasma membrane prior to virion formation.

biophysics↗

bioRxiv2019

Cysteine modification can gate non-equilibrium conformational dynamics during enzyme catalysis

Summary ParagraphProtein dynamics play an important role in enzyme catalysis1-4. Many enzymes form covalent catalytic intermediates that can alter enzyme structure and conformational dynamics5,6. How these changes in enzyme structure and dynamics facilitate passage along the reaction coordinate is a fundamental unanswered question in structural enzymology. Here, we use Mix-and-Inject Serial Femtosecond X-ray Crystallography (MISC) at an X-ray Free Electron Laser (XFEL)7-10, ambient temperature X-ray crystallography, computer simulations, and enzyme kinetics to characterize how covalent modification of the active site cysteine residue in isocyanide hydratase (ICH) alters the enzymes conformational ensemble throughout the catalytic cycle. With MISC, we directly observe formation of a thioimidate covalent intermediate during ICH catalysis. The intermediate exhibits changes in the active site electrostatic environment, disrupting a hydrogen bond and triggering a cascade of conformational changes in ICH. X-ray-induced formation of a cysteine-sulfenic acid at the catalytic nucleophile (Cys101-SOH) with conventional crystallography at ambient temperature induces similar conformational shifts, demonstrating that these enzyme motions result from cysteine modification. Computer simulations show how cysteine modification-gated structural changes allosterically propagate through the ICH dimer. Mutations at Gly150 that modulate helical mobility reduce ICH catalytic turnover and alter its pre-steady state kinetic behavior, establishing that helical mobility is important for ICH catalytic efficiency. Taken together, our results demonstrate the potential of mix-and-inject XFEL crystallography to capture otherwise elusive mechanistic details of enzyme catalysis and dynamics from microcrystalline samples7,11. This approach can connect conformational dynamics to function for the large class of systems that rely on covalently modified cysteine residues for catalysis or regulation, resolving long-standing questions about enzyme mechanism and functionally relevant non-equilibrium enzyme motions.

biochemistry↗

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↗