73 research outputs found
Testing the N-Terminal Velcro Model of CooA Carbon Monoxide Activation
No full textCooAs are dimeric bacterial CO-sensing transcription factors that activate a series of enzymes responsible for CO oxidation. The crystal structure of Rhodospirillum rubrum (rrCooA) shows that the N-terminal Pro from monomer A of the dimer coordinates the heme of monomer B that locks rrCooA in the off state. When CO binds, it is postulated that the Pro is replaced with CO, resulting in a very large reorientation of the DNA binding domains required for specific binding to DNA. Crystal structures of the closely related CooA from Carboxydothermus hydrogenoformans (chCooA) are available, and in one of these, the CO-bound on-state indicates that the N-terminal region that is displaced when CO binds provides contacts between the heme and DNA binding domains that hold the DNA binding domain in position for DNA binding. This has been termed the N-terminal velcro model of CooA activation. The study presented here tests this hypothesis by generating a disulfide mutant that covalently locks chCooA in the on-state. A simple fluorescence assay was used to measure DNA binding, and the S-S mutant was found to be in the on-state even without CO. We also determined the high-resolution crystal structure of the apo-heme domain, and the resulting structure is very similar to the holo-heme-bound structure. This result shows that the heme binding motif forms a stable structure without heme or the DNA binding domain
Abstract 4891: Identification of recurrent high-affinity MHC class I restricted neo-epitopes in neuroblastoma using ProTECT
No full textAbstract
Introduction: T-cells are trained to differentiate between cell-surface MHC-displayed peptide sequences from self- and non-self proteins and act on the latter. The numerous mutations often associated with cancers can occur in coding regions of the genome and modify the sequence of wild-type proteins, potentially creating targets for immunotherapies. We have developed an analysis pipeline ProTECT (Prediction of T-cell Epitopes for Cancer Therapy) to identify and rank neo-epitopes in terms of immunogenicity. Running ProTECT on a set of Neuroblastomas patients predicted hotspot mutations that bind well to high-frequency MHC alleles – combinations that would potentially benefit a large subset of NBL patients.
Methods: ProTECT accepts paired tumor and normal DNA sequencing fastq files, and tumor RNA sequencing fastqs. Mutations are called using a panel of callers [1-4], and are annotated[5] to identify coding mutations. Prediction of self-MHC:mutated-peptides is carried out[6] and the final binding predictions are ranked using an in-house algorithm.
Summary: Running ProTECT on 6 Neuroblastoma samples (NBL) from the TARGET (Therapeutically Applicable Research to Generate Effective Treatments) project revealed 2 well-known hotspot mutations in NBL (NRAS Q61K and ALK R1275Q) that bind to common MHC alleles (A*01:01 and B*15:01 respectively). We also found 2 closely related mutations in ALK F1174L and F1174I that are predicted to C*04:01 and C*07:02.
We carried out in-vitro refolding and crystallization assays [7] for the five highest-ranking mutant NRAS and ALK R1275Q predictions. Properly conformed MHC trimers were verified by a monodisperse peak after anion exchange chromatography. SDS gel electrophoresis and Mass-spec confirmed bound peptide for 4/5 tested predictions and 3 of these were used to set up hanging-drop crystallization trials in various conditions. Positive hits were obtained for one (AQDIYRASY::HLA-B*15:01) and the structure was obtained at 1.7A. The structure suggested the binding of the 10-mer (AQDIYRASYY) to the MHC and this was shown to bind better than the 9-mer using Differential Scanning Fluorimetry[9].
We will run ProTECT on the remaining 100+ TARGET NBL trios, and on relevant cohorts within The Cancer Genome Atlas (TCGA). We aim to reveal clinically relevant hotspot-mutation:MHC pairs.
Conclusion: We have described a pipeline for identification and ranking of therapeutically relevant neo-epitopes. We have predicted potential targets for NBL that have been validated in-vitro.
References
1. Cibulskis, K. et al. Nat Biotech 31, 213-219 (2013)
2. Radenbaugh, A. J. et al. PLoS ONE 9, e111516 (2014)
3. Larson, D. E, et.al. Bioinformatics 28 (3) (2012)
4. Saunders, C. T, et.al Bioinformatics 28 (14) (2012)
5. Cingolani, P. et al. Fly 6, 80-92 (2012)
6. Vita, R. et al. Nucl. Acids Res. (2014)
7. Garboczi, D. N. et.al. PNAS 89 (8) (1992)
9. Lance M. Hellman et.al. J Imm Meth 432:95-101 (2016)
Citation Format: Arjun A. Rao, Jugmohit Toor, Sarvind Tripathi, Jacob Pfeil, Nikolaos Sgourakis, Sofie Salama, David Haussler. Identification of recurrent high-affinity MHC class I restricted neo-epitopes in neuroblastoma using ProTECT [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4891. doi:10.1158/1538-7445.AM2017-4891</jats:p
Structural basis for tunable affinity and specificity of LxCxE-dependent protein interactions with the retinoblastoma protein family
No full textThe retinoblastoma protein (Rb) and its homologs p107 and p130 are critical regulators of gene expression during the cell cycle and are commonly inactivated in cancer. Rb proteins use their “pocket domain” to bind an LxCxE sequence motif in other proteins, many of which function with Rb proteins to co-regulate transcription. Here, we present binding data and crystal structures of the p107 pocket domain in complex with LxCxE peptides from the transcriptional co-repressor proteins HDAC1, ARID4A, and EID1. Our results explain why Rb and p107 have weaker affinity for cellular LxCxE proteins compared with the E7 protein from human papillomavirus, which has been used as the primary model for understanding LxCxE motif interactions. Our structural and mutagenesis data also identify and explain differences in Rb and p107 affinities for some LxCxE-containing sequences. Our study provides new insights into how Rb proteins bind their cell partners with varying affinity and specificity.Fil: Putta, Sivasankar. University Of California At Santa Cruz.; Estados UnidosFil: Alvarez, Lucia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Lüdtke, Stephan. No especifíca;Fil: Sehr, Peter. No especifíca;Fil: Müller, Gerd A.. University Of California At Santa Cruz.; Estados UnidosFil: Fernandez, Samantha M.. University Of California At Santa Cruz.; Estados UnidosFil: Tripathi, Sarvind. University Of California At Santa Cruz.; Estados UnidosFil: Lewis, Joe. No especifíca;Fil: Gibson, Toby J.. No especifíca;Fil: Chemes, Lucia Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Rubin, Seth M.. University Of California At Santa Cruz.; Estados Unido
Ligand and Redox Partner Binding Generates a New Conformational State in Cytochrome P450cam (CYP101A1).
No full textLigand and Redox Partner Binding Generates a New Conformational State in Cytochrome P450cam (CYP101A1)
No full textIt has become increasingly
clear that cytochromes P450 can cycle
back and forth between two extreme conformational states termed the
closed and open states. In the well-studied cytochrome P450cam, the
binding of its redox partner, putidaredoxin (Pdx), shifts P450cam
toward the open state. Shifting to the open state is thought to be
important in the formation of a proton relay network essential for
O–O bond cleavage and formation of the active Fe(IV)O
intermediate. Another important intermediate is the oxy–P450cam
complex when bound to Pdx. Trapping this intermediate in crystallo is challenging owing to its instability, but the CN– complex is both stable and an excellent mimic of the O2 complex. Here we present the P450cam–Pdx structure complexed
with CN–. CN– results in large
conformational changes including cis/trans isomerization
of proline residues. Changes include large rearrangements of active-site
residues and the formation of new active-site access channel that
we have termed channel 2. The formation of channel 2 has also been
observed in our previous molecular dynamics simulations wherein substrate
binding to an allosteric site remote from the active site opens up
channel 2. This new structure supports an extensive amount of previous
work showing that distant regions of the structure are dynamically
coupled and underscores the potentially important role that large
conformational changes and dynamics play in P450 catalysis
Structural Insights on the Conversion of Cytochrome P450 to P420
No full text[Image: see text] A characteristic feature of cytochromes P450* is that the complex formed between the ferrous heme iron and carbon monoxide generates an intense absorption band at 450 nm. This unique feature of P450s is due to the proximal thiolate Cys ligand coordinated to the heme iron. Various harsh treatments shift this band to 420 nm, thereby giving P420 which is most often associated with an inactive form of the enzyme. Various explanations have been put forward to explain the P450-to-P420 change ranging from protonation of the Cys heme ligand, displacement of the Cys ligand, or replacement of the Cys ligand with His. There are two crystal structures of the well-studied cytochrome P450cam that have a high fraction of P420. In one, P450cam is cross-linked to its redox partner, putidaredoxin (Pdx), and the second is P450cam crystallized in the absence of substrate. In both of these structures, a significant part of the substrate pocket is disordered and the poor quality of the electron density for the substrate indicates substantial disorder. However, in both structures there is no detectable change in the Cys-iron ligation or surrounding structure. These results indicate that the P450-to-P420 switch is due primarily to an opening and disordering around the substrate binding pocket and not ligand displacement or ligand swapping. Since it remains a possibility that ligand swapping could be responsible for P420 in some cases, we mutated to Gln the 3 His residues (352, 355, and 361) close enough to the proximal side of the heme that could possibly serve as heme ligands. The triple variant forms P420 which indicates that swapping Cys for His is not a requirement for the P450-to-P420 switch
Crystal structure of the Pseudomonas aeruginosa cytoplasmic heme binding protein, Apo-PhuS
No full textIron is an essential element to all living organisms and is an important determinant of bacterial virulence. Bacteria have evolved specialized systems to sequester and transport iron from the environment or host. Pseudomonas aeruginosa, an opportunistic pathogen, uses two outer membrane receptor mediated systems (Phu and Has) to utilize host heme as a source of iron. PhuS is a 39 kDa soluble cytoplasmic heme binding protein which interacts and transports heme from the inner membrane heme transporter to the cytoplasm where it is degraded by heme oxygenase thus releasing iron. PhuS is unique among other cytoplasmic heme transporter proteins owing to the presence of three histidines in the heme binding pocket which can potentially serve as heme ligands. Out of the three histidine residues on the heme binding helix, His 209 is conserved among heme trafficking proteins while His 210 and His 212 are unique to PhuS. Here we report the crystal structure of PhuS at 1.98Å resolution which shows a unique heme binding pocket and oligomeric structure compared to other known cytoplasmic heme transporter and accounts for some of the unusual biochemical properties of PhuS
Crystal Structures of Substrate-Free and Nitrosyl Cytochrome P450cin: Implications for O2 Activation
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