Engineering topics

Kreitler, Dale F.

Publications and source records attributed to Kreitler, Dale F..

9 records

DOE OSTI2024

Structural Characterization of Disulfide-Linked p53-Derived Peptide Dimers

Disulfide bonds provide a convenient method for chemoselective alteration of peptide and protein structure and function. We previously reported that mild oxidation of a p53-derived bisthiol peptide (CTFANLWRLLAQNC) under dilute non-denaturing conditions led to unexpected disulfide-linked dimers as the exclusive product. The dimers were antiparallel, significantly α-helical, resistant to protease degradation, and easily reduced back to the original bisthiol peptide. Here, in this work, we examine the intrinsic factors influencing peptide dimerization using a combination of amino acid substitution, circular dichroism (CD) spectroscopy, and X-ray crystallography. CD analysis of peptide variants suggests critical roles for Leu6 and Leu10 in the formation of stable disulfide-linked dimers. The 1.0 Å resolution crystal structure of the peptide dimer supports these data, revealing a leucine-rich LxxLL dimer interface with canonical knobs-into-holes packing. Two levels of higher-order oligomerization are also observed in the crystal: an antiparallel “dimer of dimers” mediated by Phe3 and Trp7 residues in the asymmetric unit and a tetramer of dimers mediated by Trp7 and Leu10. In CD spectra of Trp-containing peptide variants, minima at 227 nm provide evidence for the dimer of dimers in dilute aqueous solution. Importantly, and in contrast to the original dimer model, the canonical leucine-rich core and robust dimerization of most peptide variants suggests a tunable molecular architecture to target various proteins and evaluate how folding and oligomerization impact various properties, such as cell permeability.

36 MATERIALS SCIENCE↗

DOE OSTI2024

A hemoprotein with a zinc-mirror heme site ties heme availability to carbon metabolism in cyanobacteria

Abstract Heme has a critical role in the chemical framework of the cell as an essential protein cofactor and signaling molecule that controls diverse processes and molecular interactions. Using a phylogenomics-based approach and complementary structural techniques, we identify a family of dimeric hemoproteins comprising a domain of unknown function DUF2470. The heme iron is axially coordinated by two zinc-bound histidine residues, forming a distinct two-fold symmetric zinc-histidine-iron-histidine-zinc site. Together with structure-guided in vitro and in vivo experiments, we further demonstrate the existence of a functional link between heme binding by Dri1 (Domain related to iron 1, formerly ssr1698) and post-translational regulation of succinate dehydrogenase in the cyanobacterium Synechocystis , suggesting an iron-dependent regulatory link between photosynthesis and respiration. Given the ubiquity of proteins containing homologous domains and connections to heme metabolism across eukaryotes and prokaryotes, we propose that DRI (Domain Related to Iron; formerly DUF2470) functions at the molecular level as a heme-dependent regulatory domain.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2024

Activation of CBASS Cap5 endonuclease immune effector by cyclic nucleotides

The bacterial cyclic oligonucleotide-based antiphage signaling system (CBASS) is similar to the cGAS–STING system in humans, containing an enzyme that synthesizes a cyclic nucleotide on viral infection and an effector that senses the second messenger for the antiviral response. Cap5, containing a SAVED domain coupled to an HNH DNA endonuclease domain, is the most abundant CBASS effector, yet the mechanism by which it becomes activated for cell killing remains unknown. We present here high-resolution structures of full-length Cap5 from Pseudomonas syringae (Ps) with second messengers. Here, the key to PsCap5 activation is a dimer-to-tetramer transition, whereby the binding of second messenger to dimer triggers an open-to-closed transformation of the SAVED domains, furnishing a surface for assembly of the tetramer. This movement propagates to the HNH domains, juxtaposing and converting two HNH domains into states for DNA destruction. These results show how Cap5 effects bacterial cell suicide and we provide proof-in-principle data that the CBASS can be extrinsically activated to limit bacterial infections.

36 MATERIALS SCIENCE↗

DOE OSTI2023

Harnessing Aromatic‐Histidine Interactions through Synergistic Backbone Extension and Side Chain Modification

Abstract Peptide engineering efforts have delivered drugs for diverse human diseases. Side chain alteration is among the most common approaches to designing new peptides for specific applications. The peptide backbone can be modified as well, but this strategy has received relatively little attention. Here we show that new and favorable contacts between a His side chain on a target protein and an aromatic side chain on a synthetic peptide ligand can be engineered by rational and coordinated side chain modification and backbone extension. Side chain modification alone was unsuccessful. Binding measurements, high‐resolution structural studies and pharmacological outcomes all support the synergy between backbone and side chain modification in engineered ligands of the parathyroid hormone receptor‐1, which is targeted by osteoporosis drugs. These results should motivate other structure‐based designs featuring coordinated side chain modification and backbone extension to enhance the engagement of peptide ligands with target proteins.

36 MATERIALS SCIENCE↗

DOE OSTI2023

Harnessing Aromatic‐Histidine Interactions through Synergistic Backbone Extension and Side Chain Modification

36 MATERIALS SCIENCE↗

DOE OSTI2022

EstG is a novel esterase required for cell envelope integrity in Caulobacter

Proper regulation of the bacterial cell envelope is critical for cell survival. Identification and characterization of enzymes that maintain cell envelope homeostasis is crucial, as they can be targets for effective antibiotics. In this study, we have identified a novel enzyme, called EstG, whose activity protects cells from a variety of lethal assaults in the α-proteobacterium Caulobacter crescentus. Despite homology to transpeptidase family cell wall enzymes and an ability to protect against cell-wall-targeting antibiotics, EstG does not demonstrate biochemical activity toward cell wall substrates. Instead, EstG is genetically connected to the periplasmic enzymes OpgH and BglX, responsible for synthesis and hydrolysis of osmoregulated periplasmic glucans (OPGs), respectively. The crystal structure of EstG revealed similarities to esterases and transesterases, and we demonstrated esterase activity of EstG in vitro. Using biochemical fractionation, we identified a cyclic hexamer of glucose as a likely substrate of EstG. Further, this molecule is the first OPG described in Caulobacter and establishes a novel class of OPGs, the regulation and modification of which are important for stress survival and adaptation to fluctuating environments. Our data indicate that EstG, BglX, and OpgH comprise a previously unknown OPG pathway in Caulobacter. Ultimately, we propose that EstG is a novel enzyme that instead of acting on the cell wall, acts on cyclic OPGs to provide resistance to a variety of cellular stresses.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2022

AMX – the highly automated macromolecular crystallography (17-ID-1) beamline at the NSLS-II

The highly automated macromolecular crystallography beamline AMX/17-ID-1 is an undulator-based high-intensity (>5 × 10 12 photons s −1 ), micro-focus (7 µm × 5 µm), low-divergence (1 mrad × 0.35 mrad) energy-tunable (5–18 keV) beamline at the NSLS-II, Brookhaven National Laboratory, Upton, NY, USA. It is one of the three life science beamlines constructed by the NIH under the ABBIX project and it shares sector 17-ID with the FMX beamline, the frontier micro-focus macromolecular crystallography beamline. AMX saw first light in March 2016 and started general user operation in February 2017. At AMX, emphasis has been placed on high throughput, high capacity, and automation to enable data collection from the most challenging projects using an intense micro-focus beam. Here, the current state and capabilities of the beamline are reported, and the different macromolecular crystallography experiments that are routinely performed at AMX/17-ID-1 as well as some plans for the near future are presented.

36 MATERIALS SCIENCE↗

DOE OSTI2022

Hepatitis C virus NS3/4A inhibitors and other drug-like compounds as covalent binders of SARS-CoV-2 main protease

Severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2), which causes coronavirus disease 2019 (COVID-19), threatens global public health. The world needs rapid development of new antivirals and vaccines to control the current pandemic and to control the spread of the variants. Among the proteins synthesized by the SARS-CoV-2 genome, main protease (M pro also known as 3CL pro ) is a primary drug target, due to its essential role in maturation of the viral polyproteins. In this study, we provide crystallographic evidence, along with some binding assay data, that three clinically approved anti hepatitis C virus drugs and two other drug-like compounds covalently bind to the M pro Cys145 catalytic residue in the active site. Also, molecular docking studies can provide additional insight for the design of new antiviral inhibitors for SARS-CoV-2 using these drugs as lead compounds. One might consider derivatives of these lead compounds with higher affinity to the M pro as potential COVID-19 therapeutics for further testing and possibly clinical trials.

36 MATERIALS SCIENCE↗

DOE OSTI2022

A simple technique to classify diffraction data from dynamic proteins according to individual polymorphs

One often observes small but measurable differences in the diffraction data measured from different crystals of a single protein. These differences might reflect structural differences in the protein and may reveal the natural dynamism of the molecule in solution. Partitioning these mixed-state data into single-state clusters is a critical step that could extract information about the dynamic behavior of proteins from hundreds or thousands of single-crystal data sets. Mixed-state data can be obtained deliberately (through intentional perturbation) or inadvertently (while attempting to measure highly redundant single-crystal data). To the extent that different states adopt different molecular structures, one expects to observe differences in the crystals; each of the polystates will create a polymorph of the crystals. After mixed-state diffraction data have been measured, deliberately or inadvertently, the challenge is to sort the data into clusters that may represent relevant biological polystates. Here, this problem is addressed using a simple multi-factor clustering approach that classifies each data set using independent observables, thereby assigning each data set to the correct location in conformational space. This procedure is illustrated using two independent observables, unit-cell parameters and intensities, to cluster mixed-state data from chymotrypsinogen (ChTg) crystals. It is observed that the data populate an arc of the reaction trajectory as ChTg is converted into chymotrypsin.

36 MATERIALS SCIENCE↗