Cytochromes P450 (P450s) metabolize a wide range of endogenous compounds and xenobiotics, such as pollutants, environmental compounds, and drug molecules. The microsomal, membrane-associated, P450 ...isoforms CYP3A4, CYP2D6, CYP2C9, CYP2C19, CYP2E1, and CYP1A2 are responsible for the oxidative metabolism of more than 90% of marketed drugs. Cytochrome P450 3A4 (CYP3A4) metabolizes more drug molecules than all other isoforms combined. Here we report three crystal structures of CYP3A4: unliganded, bound to the inhibitor metyrapone, and bound to the substrate progesterone. The structures revealed a surprisingly small active site, with little conformational change associated with the binding of either compound. An unexpected peripheral binding site is identified, located above a phenylalanine cluster, which may be involved in the initial recognition of substrates or allosteric effectors.
The ability of electrospray to propel large viruses into a mass spectrometer is established and is rationalized by analogy to the atmospheric transmission of the common cold. Much less clear is the ...fate of membrane-embedded molecular machines in the gas phase. Here we show that rotary adenosine triphosphatases (ATPases)/synthases from Thermus thermophilus and Enterococcus hirae can be maintained intact with membrane and soluble subunit interactions preserved in vacuum. Mass spectra reveal subunit stoichiometries and the identity of tightly bound lipids within the membrane rotors. Moreover, subcomplexes formed in solution and gas phases reveal the regulatory effects of nucleotide binding on both ATP hydrolysis and proton translocation. Consequently, we can link specific lipid and nucleotide binding with distinct regulatory roles.
The transcription factor FOXM1 binds to sequence-specific motifs on DNA (C/TAAACA) through its DNA-binding domain (DBD) and activates proliferation- and differentiation-associated genes. Aberrant ...overexpression of FOXM1 is a key feature in oncogenesis and progression of many human cancers. Here--from a high-throughput screen applied to a library of 54,211 small molecules--we identify novel small molecule inhibitors of FOXM1 that block DNA binding. One of the identified compounds, FDI-6 (NCGC00099374), is characterized in depth and is shown to bind directly to FOXM1 protein, to displace FOXM1 from genomic targets in MCF-7 breast cancer cells, and induce concomitant transcriptional downregulation. Global transcript profiling of MCF-7 cells by RNA-seq shows that FDI-6 specifically downregulates FOXM1-activated genes with FOXM1 occupancy confirmed by ChIP-PCR. This small molecule-mediated effect is selective for FOXM1-controlled genes with no effect on genes regulated by homologous forkhead family factors.
Given the frequent use of DMSO in biochemical and biophysical assays, it is desirable to understand the influence of DMSO concentration on the dissociation or unfolding behavior of proteins. In this ...study, the effects of DMSO on the structure and interactions of avidin and Mycobacterium tuberculosis (Mtb) CYP142A1 were assessed through collision-induced dissociation (CID) and collision-induced unfolding (CIU) as monitored by nanoelectrospray ionization–ion mobility–mass spectrometry (nESI-IM-MS). DMSO concentrations higher than 4% (v/v) destabilize the avidin tetramer toward dissociation and unfolding, via both its effects on charge state distribution (CSD) as well as at the level of individual charge states. In contrast, DMSO both protects against heme loss and increases the stability of CYP142A1 toward unfolding even up to 40% DMSO. Tandem MS/MS experiments showed that DMSO could modify the dissociation pathway of CYP142A1, while CIU revealed the protective effect of the heme group on the structure of CYP142A1.
Proteomic studies have yielded detailed lists of protein components. Relatively little is known, however, of interactions between proteins or of their spatial arrangement. To bridge this gap, we are ...developing a mass spectrometry approach based on intact protein complexes. By studying intact complexes, we show that we are able to not only determine the stoichiometry of all subunits present but also deduce interaction maps and topological arrangements of subunits. To construct an interaction network, we use tandem mass spectrometry to define peripheral subunits and partial denaturation in solution to generate series of subcomplexes. These subcomplexes are subsequently assigned using tandem mass spectrometry. To facilitate this assignment process, we have developed an iterative search algorithm (SUMMIT) to both assign protein subcomplexes and generate protein interaction networks. This software package not only allows us to construct the subunit architecture of protein assemblies but also allows us to explore the limitations and potential of our approach. Using series of hypothetical complexes, generated at random from protein assemblies containing between six and fourteen subunits, we highlight the significance of tandem mass spectrometry for defining subunits present. We also demonstrate the importance of pairwise interactions and the optimal numbers of subcomplexes required to assign networks with up to fourteen subunits. To illustrate application of our approach, we describe the overall architecture of two endogenous protein assemblies isolated from yeast at natural expression levels, the 19S proteasome lid and the RNA exosome. In constructing our models, we did not consider previous electron microscopy images but rather deduced the subunit architecture from series of subcomplexes and our network algorithm. The results show that the proteasome lid complex consists of a bicluster with two tetrameric lobes. The exosome lid, by contrast, is a six-membered ring with three additional bridging subunits that confer stability to the ring and with a large subunit located at the base. Significantly, by combining data from MS and homology modeling, we were able to construct an atomic model of the yeast exosome. In summary, the architectural and atomic models of both protein complexes described here have been produced in advance of high-resolution structural data and as such provide an initial model for testing hypotheses and planning future experiments. In the case of the yeast exosome, the atomic model is validated by comparison with the atomic structure from X-ray diffraction of crystals of the reconstituted human exosome, which is homologous to that of the yeast. Overall therefore this mass spectrometry and homology modeling approach has given significant insight into the structure of two previously intractable protein complexes and as such has broad application in structural biology.
Efficient duplication of the genome requires the concerted action of helicase and DNA polymerases at replication forks to avoid stalling of the replication machinery and consequent genomic ...instability. In eukaryotes, the physical coupling between helicase and DNA polymerases remains poorly understood. Here we define the molecular mechanism by which the yeast Ctf4 protein links the Cdc45-MCM-GINS (CMG) DNA helicase to DNA polymerase α (Pol α) within the replisome. We use X-ray crystallography and electron microscopy to show that Ctf4 self-associates in a constitutive disk-shaped trimer. Trimerization depends on a β-propeller domain in the carboxy-terminal half of the protein, which is fused to a helical extension that protrudes from one face of the trimeric disk. Critically, Pol α and the CMG helicase share a common mechanism of interaction with Ctf4. We show that the amino-terminal tails of the catalytic subunit of Pol α and the Sld5 subunit of GINS contain a conserved Ctf4-binding motif that docks onto the exposed helical extension of a Ctf4 protomer within the trimer. Accordingly, we demonstrate that one Ctf4 trimer can support binding of up to three partner proteins, including the simultaneous association with both Pol α and GINS. Our findings indicate that Ctf4 can couple two molecules of Pol α to one CMG helicase within the replisome, providing a new model for lagging-strand synthesis in eukaryotes that resembles the emerging model for the simpler replisome of Escherichia coli. The ability of Ctf4 to act as a platform for multivalent interactions illustrates a mechanism for the concurrent recruitment of factors that act together at the fork.
Native nanoelectrospray ionization mass spectrometry is an underutilized technique for fragment screening. In this study, the first demonstration is provided of the use of native mass spectrometry ...for screening fragments against a protein–DNA interaction. EthR is a transcriptional repressor of EthA expression in Mycobacterium tuberculosis (Mtb) that reduces the efficacy of ethionamide, a second‐line antitubercular drug used to combat multidrug‐resistant Mtb strains. A small‐scale fragment screening campaign was conducted against the EthR–DNA interaction using native mass spectrometry, and the results were compared with those from differential scanning fluorimetry, a commonly used primary screening technique. Hits were validated by surface plasmon resonance and X‐ray crystallography. The screening campaign identified two new fragments that disrupt the EthR–DNA interaction in vitro (IC50=460–610 μm) and bind to the hydrophobic channel of the EthR dimer.
Native mass spectrometry has been applied for fragment screening against a protein–DNA interaction for the first time. Two new fragments were thus identified. They were validated and their binding was characterized against EthR by native MS, surface plasmon resonance, and X‐ray crystallography.
CK2 is an intrinsically active protein kinase that is crucial for cellular viability. However, conventional kinase regulatory mechanisms do not apply to CK2, and its mode of regulation remains ...elusive. Interestingly, CK2 is known to undergo reversible ionic-strength-dependent oligomerization. Furthermore, a regulatory mechanism based on autoinhibitory oligomerization has been postulated on the basis of the observation of circular trimeric oligomers and linear CK2 assemblies in various crystal structures. Here, we employ native mass spectrometry to monitor the assembly of oligomeric CK2 species in an ionic-strength-dependent manner. A subsequent combination of ion mobility spectrometry–mass spectrometry and hydrogen–deuterium exchange mass spectrometry techniques was used to analyze the conformation of CK2 oligomers. Our findings support ionic-strength-dependent CK2 oligomerization, demonstrate the transient nature of the α/β interaction, and show that CK2 oligomerization proceeds via both the circular and linear assembly.
The maintenance of genomic stability relies on the spindle assembly checkpoint (SAC), which ensures accurate chromosome segregation by delaying the onset of anaphase until all chromosomes are ...properly bioriented and attached to the mitotic spindle. BUB1 and BUBR1 kinases are central for this process and by interacting with Blinkin, link the SAC with the kinetochore, the macromolecular assembly that connects microtubules with centromeric DNA. Here, we identify the Blinkin motif critical for interaction with BUBR1, define the stoichiometry and affinity of the interaction, and present a 2.2 Å resolution crystal structure of the complex. The structure defines an unanticipated BUBR1 region responsible for the interaction and reveals a novel Blinkin motif that undergoes a disorder-to-order transition upon ligand binding. We also show that substitution of several BUBR1 residues engaged in binding Blinkin leads to defects in the SAC, thus providing the first molecular details of the recognition mechanism underlying kinetochore-SAC signaling.
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► Molecular details of the recognition mechanism underlying kinetochore-SAC signalling ► Crystal structure of a BUBR1-Blinkin complex defines an unanticipated BUBR1 region ► A novel Blinkin motif undergoes a disorder-to-order transition upon BUBR1 binding ► Mutation of BUBR1 residues that bind Blinkin lead to an impaired mitotic checkpoint
Nondenaturing nanoelectrospray ionization mass spectrometry (nanoESI MS) of intact protein complexes was used to study CYP121, one of the 20 cytochrome P450s in Mycobacterium tuberculosis (Mtb) and ...an enzyme that is essential for bacterial viability. The results shed new light on both ligand-free and ligand-bound states of CYP121. Isolated unbound CYP121 is a predominantly dimeric protein, with a minor monomeric form present. High affinity azoles cause the dissociation of dimeric CYP121 into monomer, whereas weaker azole binders induce partial dimer dissociation or do not significantly destabilize the dimer. Complexes of CYP121 with azoles were poorly detected by nanoESI MS, indicating kinetically labile complexes that are easily prone to gas-phase dissociation. Unlike with the azoles, CYP121 forms a stable complex with its natural substrate cYY that does not undergo gas-phase dissociation. In addition, a series of potential ligands from fragment-based studies were used as a test for nanoESI MS work against CYP121. Most of these ligands formed stable complexes with CYP121, and their binding did not promote dimer dissociation. On the basis of binding to the monomer and/or CYP121 dimer it was possible to determine the relative order of their CYP121 binding affinities. The top nanoESI MS screening hit was confirmed by heme absorbance shift assay to have a K d of 40 μM.
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