NMRFAM-SPARKY: enhanced software for biomolecular NMR spectroscopy

Background:The RING E3 AO7/RNF25 binds its E2 with unusually high affinity. Results: AO7 has a secondary E2 binding site adjacent to the RING. Conclusion: This site prevents the stimulatory effect of non-covalent backside binding of ubiquitin, has unique agonist properties, and allows for structural analysis of RING mutants. Significance: Knowledge of how RING E3s mediate ubiquitination is critical to understanding cellular protein regulation.

Section: Methodsmentioning

confidence: 99%

Insights into Ubiquitination from the Unique Clamp-like Binding of the RING E3 AO7 to the E2 UbcH5B

Liang

Mariano

et al. 2015

“…The programs istHMS (27) and NMRPipe (20) were used to reconstruct and process three-dimensional NUS data. Processed spectra were analyzed using NMRFAMSparky (28). Backbone assignments were made automatically with the PINE-NMR software (29) and confirmed manually using NMRFAM-Sparky (28 …”

Section: Methodsmentioning

confidence: 99%

“…Processed spectra were analyzed using NMRFAMSparky (28). Backbone assignments were made automatically with the PINE-NMR software (29) and confirmed manually using NMRFAM-Sparky (28 …”

Section: Methodsmentioning

confidence: 99%

Defining a Two-pronged Structural Model for PB1 (Phox/Bem1p) Domain Interaction in Plant Auxin Responses

Korasick

Chatterjee

Tonelli

et al. 2015

Self Cite

Background: Phox/Bem1p domains are universal domains that organize cellular signaling scaffolds. Results: Biophysical analyses reveal driving forces and core residues involved in PB1 interaction. Conclusion: Electrostatic interactions focused around two complementary prongs. Significance: These results provide the first in-depth analysis of the factors driving self-interaction of a type I/II PB1 domain.

“…Data were processed using NMRPipe (54), with the SMILE algorithm used for prediction of the missing points in the 13 C and 15 N dimensions of the non-uniform sampling data sets (55). Data analysis was performed using NMRFAM-SPARKY (56).…”

Section: Nmr Spectroscopymentioning

confidence: 99%

An engineered transforming growth factor β (TGF-β) monomer that functions as a dominant negative to block TGF-β signaling

Kim¹,

Barron²,

Hinck

et al. 2017

Edited by Norma AllewellThe transforming growth factor ␤ isoforms, TGF-␤1, -␤2, and -␤3, are small secreted homodimeric signaling proteins with essential roles in regulating the adaptive immune system and maintaining the extracellular matrix. However, dysregulation of the TGF-␤ pathway is responsible for promoting the progression of several human diseases, including cancer and fibrosis. Despite the known importance of TGF-␤s in promoting disease progression, no inhibitors have been approved for use in humans. Herein, we describe an engineered TGF-␤ monomer, lacking the heel helix, a structural motif essential for binding the TGF-␤ type I receptor (T␤RI) but dispensable for binding the other receptor required for TGF-␤ signaling, the TGF-␤ type II receptor (T␤RII), as an alternative therapeutic modality for blocking TGF-␤ signaling in humans. As shown through binding studies and crystallography, the engineered monomer retained the same overall structure of native TGF-␤ monomers and bound T␤RII in an identical manner. Cell-based luciferase assays showed that the engineered monomer functioned as a dominant negative to inhibit TGF-␤ signaling with a K i of 20 -70 nM. Investigation of the mechanism showed that the high affinity of the engineered monomer for T␤RII, coupled with its reduced ability to non-covalently dimerize and its inability to bind and recruit T␤RI, enabled it to bind endogenous T␤RII but prevented it from binding and recruiting T␤RI to form a signaling complex. Such engineered monomers provide a new avenue to probe and manipulate TGF-␤ signaling and may inform similar modifications of other TGF-␤ family members.The transforming growth factor ␤ isoforms, TGF-␤1, -␤2, and -␤3, are small secreted signaling proteins. Their overall structures are similar and consist of two cystine-knotted monomers tethered together by a single inter-chain disulfide bond (1). They coordinate wound healing, modulate immune cell function, maintain the extracellular matrix, and regulate epithelial and endothelial cell growth and differentiation (2). The TGF-␤s are synthesized as pre-pro-proteins, and after maturation, secretion, and release from their pro-domains (3), the mature homodimeric growth factors (GFs) 3 bind and bring together two single-pass transmembrane receptors, known as T␤RI and T␤RII, to form the signaling-competent T␤RI 2 -T␤RII 2 heterotetramer (4, 5). TGF-␤ GFs assemble T␤RI 2 -T␤RII 2 heterotetramer in a sequential manner, first by binding T␤RII followed by recruitment of T␤RI (6, 7). The stepwise assembly of T␤RII and T␤RI into a heterotetramer is driven by binding of T␤RI to a composite TGF-␤/T␤RII interface (Fig. 1A) (8, 9).The disruption or dysregulation of the TGF-␤ pathway is responsible for several human diseases. These include connec-