Protein conformations are shaped by cellular environments, but how environmental changes alter the conformational landscapes of specific proteins in vivo remains largely uncharacterized, in part due ...to the challenge of probing protein structures in living cells. Here, we use deep mutational scanning to investigate how a toxic conformation of α-synuclein, a dynamic protein linked to Parkinson’s disease, responds to perturbations of cellular proteostasis. In the context of a course for graduate students in the UCSF Integrative Program in Quantitative Biology, we screened a comprehensive library of α-synuclein missense mutants in yeast cells treated with a variety of small molecules that perturb cellular processes linked to α-synuclein biology and pathobiology. We found that the conformation of α-synuclein previously shown to drive yeast toxicityan extended, membrane-bound helixis largely unaffected by these chemical perturbations, underscoring the importance of this conformational state as a driver of cellular toxicity. On the other hand, the chemical perturbations have a significant effect on the ability of mutations to suppress α-synuclein toxicity. Moreover, we find that sequence determinants of α-synuclein toxicity are well described by a simple structural model of the membrane-bound helix. This model predicts that α-synuclein penetrates the membrane to constant depth across its length but that membrane affinity decreases toward the C terminus, which is consistent with orthogonal biophysical measurements. Finally, we discuss how parallelized chemical genetics experiments can provide a robust framework for inquiry-based graduate coursework.
Metastatic UTUC is an aggressive disease. 45 patients with distant metastasis were analysed at the time of initiating chemotherapy in a risk score that includes anemia and receipt of cisplatin ...helping stratify overall survival patients for future clinical trials.
Upper tract urothelial carcinomas (UTUCs) are increasingly recognized as separate malignancies. Additional insight into clinical outcomes and key prognostic factors are needed.
To detail outcomes of patients with UTUCs recurring after radical nephroureterectomy (RNU) and to determine a risk score that predicts outcomes of patients with non–lymph node distant metastasis.
Chart review of all patients who had an extraurothelial recurrence after RNU for UTUC at Dana-Farber Cancer Institute between 2009 and 2014.
Median overall survival defined as time from chemotherapy for distant relapse to death. Prognostic relevance of performance status, hemoglobin, and receipt of cisplatin were assessed by Cox regression model.
A total of 102 patients were identified, 57 of whom had non–lymph node distant metastases at relapse; 45 received chemotherapy. Median follow-up was 29.8 months; median overall survival was 14.7 months. Objective response rate to any chemotherapy in the first-line setting was only 22%. Hemoglobin > 11 g/dL and receipt of cisplatin was associated with numerically longer median survival but did not reach statistical significance in univariate and multivariate analysis. Prognostic risk score scale including hemoglobin < 11 g/dL and receipt of cisplatin was inversely associated with survival, with scores of 0, 1, and 2 leading to median survival of 19.0, 14.9, and 7.2 months (P = .38), respectively.
Advanced UTUC portends a poor prognosis, and most patients cannot receive cisplatin-based chemotherapy. A risk score that includes anemia and receipt of cisplatin helps stratify patients with distant metastasis for inclusion into eventual clinical trials. More studies are needed to validate these findings.
Metastatic UTUC is an aggressive disease, where anemia and ineligibility to receive cisplatin are adverse features associated with shorter survival.
Conformational ensembles underlie all protein functions. Thus, acquiring atomic-level ensemble models that accurately represent conformational heterogeneity is vital to deepen our understanding of ...how proteins work. Modeling ensemble information from X-ray diffraction data has been challenging, as traditional cryo-crystallography restricts conformational variability while minimizing radiation damage. Recent advances have enabled the collection of high quality diffraction data at ambient temperatures, revealing innate conformational heterogeneity and temperature-driven changes. Here, we used diffraction datasets for Proteinase K collected at temperatures ranging from 313 to 363 K to provide a tutorial for the refinement of multiconformer ensemble models. Integrating automated sampling and refinement tools with manual adjustments, we obtained multiconformer models that describe alternative backbone and sidechain conformations, their relative occupancies, and interconnections between conformers. Our models revealed extensive and diverse conformational changes across temperature, including increased bound peptide ligand occupancies, different Ca
binding site configurations and altered rotameric distributions. These insights emphasize the value and need for multiconformer model refinement to extract ensemble information from diffraction data and to understand ensemble-function relationships.
In their folded state, biomolecules exchange between multiple conformational states that are crucial for their function. Traditional structural biology methods, such as X-ray crystallography and ...cryogenic electron microscopy (cryo-EM), produce density maps that are ensemble averages, reflecting molecules in various conformations. Yet, most models derived from these maps explicitly represent only a single conformation, overlooking the complexity of biomolecular structures. To accurately reflect the diversity of biomolecular forms, there is a pressing need to shift toward modeling structural ensembles that mirror the experimental data. However, the challenge of distinguishing signal from noise complicates manual efforts to create these models. In response, we introduce the latest enhancements to qFit, an automated computational strategy designed to incorporate protein conformational heterogeneity into models built into density maps. These algorithmic improvements in qFit are substantiated by superior R free and geometry metrics across a wide range of proteins. Importantly, unlike more complex multicopy ensemble models, the multiconformer models produced by qFit can be manually modified in most major model building software (e.g., Coot) and fit can be further improved by refinement using standard pipelines (e.g., Phenix, Refmac, Buster). By reducing the barrier of creating multiconformer models, qFit can foster the development of new hypotheses about the relationship between macromolecular conformational dynamics and function.
Protein folding converts a disordered polymer to a globular structure, reducing many conformational degrees of freedom and incurring a significant conformational entropy penalty. However, residual ...conformational entropy is retained in a protein’s folded native state. Subtle changes between positions within the native state, mostly from sidechains, alters residual conformational entropy, leading to differences in binding affinity and allosteric communication. While NMR has provided measurements of conformational entropy, these measurements do not provide information on where this entropy is coming from, such as if this is coming from a sidechain moving harmonically or anharmonically. However, we can take advantage of the fact that X-ray crystallography and CryoEM experimental data capture the conformational ensemble allowing us to measure the motion of residues and their atomistic structure. This provides an unparalleled platform to answer how, where, and why conformational entropy. The first chapter of this thesis presents the improvements to the qFit algorithm. This algorithm allows for the automated modeling of multiple conformations per residue across a protein for high resolution X-ray crystallography or cryo-EM. We present algorithm improvements including the ability to run the program on a laptop. This algorithm was the basis for much of the future work of my thesis.The second chapter contains the findings of the relationship between conformational heterogeneity and ligand binding. Using qFit, we identified the changes in conformational heterogeneity between matched bound and unbound high resolution X-ray structures. We identified a reciprocal relationship upon ligand binding where as binding site residues become more rigid, distant residues become more flexible, indicating an entropic compensation. The third chapter contains my future outlook on the questions and techniques to probe conformational entropy mechanism. This chapter includes how to integrate new modeling techniques to understand how different motions of residues lead to differences in conformational entropy.
Immune checkpoint inhibitors targeting the programmed cell death 1 receptor (PD-1) improve survival in a subset of patients with clear cell renal cell carcinoma (ccRCC). To identify genomic ...alterations in ccRCC that correlate with response to anti-PD-1 monotherapy, we performed whole-exome sequencing of metastatic ccRCC from 35 patients. We found that clinical benefit was associated with loss-of-function mutations in the
gene (
= 0.012), which encodes a subunit of the PBAF switch-sucrose nonfermentable (SWI/SNF) chromatin remodeling complex. We confirmed this finding in an independent validation cohort of 63 ccRCC patients treated with PD-1 or PD-L1 (PD-1 ligand) blockade therapy alone or in combination with anti-CTLA-4 (cytotoxic T lymphocyte-associated protein 4) therapies (
= 0.0071). Gene-expression analysis of PBAF-deficient ccRCC cell lines and
-deficient tumors revealed altered transcriptional output in JAK-STAT (Janus kinase-signal transducers and activators of transcription), hypoxia, and immune signaling pathways.
loss in ccRCC may alter global tumor-cell expression profiles to influence responsiveness to immune checkpoint therapy.
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