CRISPR–Cas9 is a widely employed genome-editing tool with functionality reliant on the ability of the Cas9 endonuclease to introduce site-specific breaks in double-stranded DNA. In this system, an ...intriguing allosteric communication has been suggested to control its DNA cleavage activity through flexibility of the catalytic HNH domain. Here, solution NMR experiments and a novel Gaussian-accelerated molecular dynamics (GaMD) simulation method are used to capture the structural and dynamic determinants of allosteric signaling within the HNH domain. We reveal the existence of a millisecond time scale dynamic pathway that spans HNH from the region interfacing the adjacent RuvC nuclease and propagates up to the DNA recognition lobe in full-length CRISPR–Cas9. These findings reveal a potential route of signal transduction within the CRISPR–Cas9 HNH nuclease, advancing our understanding of the allosteric pathway of activation. Further, considering the role of allosteric signaling in the specificity of CRISPR–Cas9, this work poses the mechanistic basis for novel engineering efforts aimed at improving its genome-editing capability.
Identifying structural differences among proteins can be a non-trivial task. When contrasting ensembles of protein structures obtained from molecular dynamics simulations, biologically-relevant ...features can be easily overshadowed by spurious fluctuations. Here, we present SINATRA Pro, a computational pipeline designed to robustly identify topological differences between two sets of protein structures. Algorithmically, SINATRA Pro works by first taking in the 3D atomic coordinates for each protein snapshot and summarizing them according to their underlying topology. Statistically significant topological features are then projected back onto a user-selected representative protein structure, thus facilitating the visual identification of biophysical signatures of different protein ensembles. We assess the ability of SINATRA Pro to detect minute conformational changes in five independent protein systems of varying complexities. In all test cases, SINATRA Pro identifies known structural features that have been validated by previous experimental and computational studies, as well as novel features that are also likely to be biologically-relevant according to the literature. These results highlight SINATRA Pro as a promising method for facilitating the non-trivial task of pattern recognition in trajectories resulting from molecular dynamics simulations, with substantially increased resolution.
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Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
MIF is a ubiquitous protein involved in proinflammatory processes, which undergoes an oxidation-driven conformational change to oxidized (ox)MIF. We demonstrate that hypochlorous acid, produced by ...neutrophil-released myeloperoxidase (MPO) under inflammatory conditions, effectively oxidizes MIF into the oxMIF isoform, which is specifically recognized by the anti-oxMIF therapeutic antibody, ON104. NMR investigation of MIF oxidized by the MPO system revealed increased flexibility throughout the MIF structure, including at several catalytic and allosteric sites. Mass spectrometry of MPO-oxMIF revealed methionines as the primary site of oxidation, whereas Pro2 and Tyr99/100 remained almost unmodified. ELISA, SPR and cell-based assays demonstrated that structural changes caused by MPO-driven oxidation promoted binding of oxMIF to its receptor, CD74, which does not occur with native MIF. These data reveal the environment and modifications that facilitate interactions between MIF and its pro-inflammatory receptor, and a route for therapeutic intervention targeting the oxMIF isoform.
Allostery is a ubiquitous biological mechanism in which a distant binding site is coupled to and drastically alters the function of a catalytic site in a protein. Allostery provides a high level of ...spatial and temporal control of the integrity and activity of biomolecular assembles composed of proteins, nucleic acids, or small molecules. Understanding the physical forces that drive allosteric coupling is critical to harnessing this process for use in bioengineering, de novo protein design, and drug discovery. Current microscopic models of allostery highlight the importance of energetics, structural rearrangements, and conformational fluctuations, and in this review, we discuss the synergistic use of solution NMR spectroscopy and computational methods to probe these phenomena in allosteric systems, particularly protein-nucleic acid complexes. This combination of experimental and theoretical techniques facilitates an unparalleled detection of subtle changes to structural and dynamic equilibria in biomolecules with atomic resolution, and we provide a detailed discussion of specialized NMR experiments as well as the complementary methods that provide valuable insight into allosteric pathways in silico. Lastly, we highlight two case studies to demonstrate the adaptability of this approach to enzymes of varying size and mechanistic complexity.
The CRISPR-associated protein 9 (Cas9) has been engineered as a precise gene editing tool to make double-strand breaks. CRISPR-associated protein 9 binds the folded guide RNA (gRNA) that serves as a ...binding scaffold to guide it to the target DNA duplex
a RecA-like strand-displacement mechanism but without ATP binding or hydrolysis. The target search begins with the protospacer adjacent motif or PAM-interacting domain, recognizing it at the major groove of the duplex and melting its downstream duplex where an RNA-DNA heteroduplex is formed at nanomolar affinity. The rate-limiting step is the formation of an R-loop structure where the HNH domain inserts between the target heteroduplex and the displaced non-target DNA strand. Once the R-loop structure is formed, the non-target strand is rapidly cleaved by RuvC and ejected from the active site. This event is immediately followed by cleavage of the target DNA strand by the HNH domain and product release. Within CRISPR-associated protein 9, the HNH domain is inserted into the RuvC domain near the RuvC active site
two linker loops that provide allosteric communication between the two active sites. Due to the high flexibility of these loops and active sites, biophysical techniques have been instrumental in characterizing the dynamics and mechanism of the CRISPR-associated protein 9 nucleases, aiding structural studies in the visualization of the complete active sites and relevant linker structures. Here, we review biochemical, structural, and biophysical studies on the underlying mechanism with emphasis on how CRISPR-associated protein 9 selects the target DNA duplex and rejects non-target sequences.
Macrophage migration inhibitory factor (MIF) is an inflammatory protein with various non-overlapping functions. It is not only conserved in mammals, but it is found in parasites, fish, and plants. ...Human MIF is a homotrimer with an enzymatic cavity between two subunits with Pro1 as a catalytic base, activates the receptors CD74, CXCR2, and CXCR4, has functional interactions in the cytosol, and is reported to be a nuclease. There is a solvent channel down its 3-fold axis with a recently identified gating residue as an allosteric site important for regulating, to different extents, the enzymatic activity and CD74 binding and signaling. In this study we explore the consequence of converting the allosteric residue Tyr99 to cysteine (Y99C) and characterize its crystallographic structure, NMR dynamics, stability, CD74 function, and enzymatic activity. In addition to the homotrimeric variant, we develop strategies for expressing and purifying a heterotrimeric variant consisting of mixed wild type and Y99C for characterization of the allosteric site to provide more insight.
CXCL12 activates CXCR4 and is involved in embryogenesis, hematopoiesis, and angiogenesis. It has pathological roles in HIV-1, WHIM disease, cancer, and autoimmune diseases. An antagonist, AMD3100, is ...used for the release of CD34+ hematopoietic stem cells from the bone marrow for autologous transplantation for lymphoma or multiple myeloma patients. Adverse effects are tolerated due to its short-term treatment, but AMD3100 is cardiotoxic in clinical studies for HIV-1. In an effort to determine whether
Saccharomyces cerevisiae
expressing a functional human CXCR4 could be used as a platform for identifying a ligand from a library of less ∼1,000 compounds, a high-throughput screening was developed. We report that 2-carboxyphenyl phosphate (fosfosal) up-regulates CXCR4 activation only in the presence of CXCL12. This is the first identification of a compound that increases CXCR4 activity by any mechanism. We mapped the fosfosal binding site on CXCL12, described its mechanism of action, and studied its chemical components, salicylate and phosphate, to conclude that they synergize to achieve the functional effect.
•Analyses of NMR chemical shifts are described for the elucidation of allosteric coupling within proteins.•A focus on the quintessential simple readout of NMR spectroscopy, the chemical shift, ...provides an opportunity for high levels of insight for specialists and non-specialists.•Practical considerations for various analyses are discussed, as is the complementarity between various methods.•Protein conformational equilibria are dissected on multiple exchange regimes to reveal networks of amino acids that drive functional responses.
The exquisite sensitivity of the NMR chemical shift to local environment makes it an ideal probe to assess atomic level perturbations in proteins of all sizes and structural compositions. Recent advances in solution and solid-state NMR spectroscopy of biomolecules have leveraged the chemical shift to report on short- and long-range couplings between individual amino acids to establish “networks” of residues that form the basis of allosteric pathways that transmit chemical signals through the protein matrix to induce functional responses. The simple premise that thermodynamically and functionally coupled regions of a protein (i.e. active and allosteric sites) should be reciprocally sensitive to structural or dynamic perturbations has enabled NMR spectroscopy, the premier method for molecular resolution of protein structural fluctuations, to occupy a place at the forefront of investigations into protein allostery. Here, we detail several key methods of NMR chemical shift analysis to extract mechanistic information about long-range chemical signaling in a protein, focusing on practical methodological aspects and the circumstances under which a given approach would be relevant. We also detail some of the experimental considerations that should be made when applying these methods to specific protein systems.
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