scholarly journals ANTH domains within CALM, HIP1R, and Sla2 recognize ubiquitin internalization signals.

2021 ◽  
Author(s):  
Natalya Pashkova ◽  
Lokesh Gakhar ◽  
Stanley Winistorfer ◽  
Annabel Y Minard ◽  
Nicholas J Schnicker ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Membrane Proteins ◽  
Binding Proteins ◽  
Terminal Region ◽  
Regulatory Mechanisms ◽  
Solution Nmr ◽  
Nmr Studies ◽  
Binding Domains ◽  
Binding Regions

Ubiquitin (Ub) serves as a signal for clathrin-mediated endocytosis (CME) by engaging Ub-binding proteins with the internalization apparatus. Ub is a versatile internalization signal because it can be added to a wide variety of membrane proteins, expanding the capacity of cells to use a variety of regulatory mechanisms to specify the conditions under which a particular protein will be internalized. Several candidate adaptors that can recognize ubiquitinated membrane proteins have been identified that work in endocytic processes that are both clathrin-dependent and independent. These include Epsin and Eps15, which bind and help sort Ub-cargo into internalization sites. Here we identify additional components of the endocytosis apparatus that bind Ub. The N-terminal ANTH domains found in CALM, AP180, HIP1R and yeast Sla2 all bind monoubiquitin with micromolar affinity. ANTH domains belong to a larger superfamily of domains including ENTH and VHS domains, many of which have Ub-binding regions outside of their VHS/ENTH/ANTH domains that enable them to mediate Ub-dependent sorting events throughout the cell. Solution NMR studies combined with a crystal structure of the CALM ANTH domain in a complex with Ub show that Ub binds to a C-terminal region of the ANTH domain that is not present in ENTH domains. Combined loss of Ub-binding by ANTH-domain proteins and other Ub-binding domains within the internalization apparatus of yeast caused defects in the Ub-dependent internalization of the GPCR Ste2 but had no effect on internalization of Ste2 via other internalization signals. These studies define new components of the internalization machinery that work collectively with Epsin and Eps15 to specify recognition of Ub as an internalization signal.

Synthetic Biology-Based Solution NMR Studies on Membrane Proteins in Lipid Environments

2019 ◽  
pp. 143-185 ◽  
Author(s):  
Erik Henrich ◽  
Frank Löhr ◽  
Julija Mezhyrova ◽  
Aisha Laguerre ◽  
Frank Bernhard ◽  
...  
Keyword(s):  
Synthetic Biology ◽  
Membrane Proteins ◽  
Solution Nmr ◽  
Nmr Studies

scholarly journals An Adaptable Phospholipid Membrane Mimetic System for Solution NMR Studies of Membrane Proteins

2017 ◽  
Vol 139 (42) ◽  
pp. 14829-14832 ◽  
Author(s):  
Chih-Ta Henry Chien ◽  
Lukas R. Helfinger ◽  
Mark J. Bostock ◽  
Andras Solt ◽  
Yi Lei Tan ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Solution Nmr ◽  
Phospholipid Membrane ◽  
Nmr Studies ◽  
Membrane Mimetic

The Use of Amphipols for Solution NMR Studies of Membrane Proteins: Advantages and Constraints as Compared to Other Solubilizing Media

2014 ◽  
Vol 247 (9-10) ◽  
pp. 827-842 ◽  
Author(s):  
Noelya Planchard ◽  
Élodie Point ◽  
Tassadite Dahmane ◽  
Fabrice Giusti ◽  
Marie Renault ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Solution Nmr ◽  
Nmr Studies

scholarly journals A noncanonical PWI domain in the N-terminal helicase-associated region of the spliceosomal Brr2 protein

2015 ◽  
Vol 71 (4) ◽  
pp. 762-771 ◽  
Author(s):  
Eva Absmeier ◽  
Leonie Rosenberger ◽  
Luise Apelt ◽  
Christian Becke ◽  
Karine F. Santos ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Nucleic Acid ◽  
Messenger Rna ◽  
Spectroscopic Studies ◽  
Terminal Region ◽  
Surface Patch ◽  
Molecular Architecture ◽  
Nucleic Acid Binding ◽  
Band Shift ◽  
Binding Domains

The spliceosomal RNA helicase Brr2 is required for the assembly of a catalytically active spliceosome on a messenger RNA precursor. Brr2 exhibits an unusual organization with tandem helicase units, each comprising dual RecA-like domains and a Sec63 homology unit, preceded by a more than 400-residue N-terminal helicase-associated region. Whereas recent crystal structures have provided insights into the molecular architecture and regulation of the Brr2 helicase region, little is known about the structural organization and function of its N-terminal part. Here, a near-atomic resolution crystal structure of a PWI-like domain that resides in the N-terminal region ofChaetomium thermophilumBrr2 is presented. CD spectroscopic studies suggested that this domain is conserved in the yeast and human Brr2 orthologues. Although canonical PWI domains act as low-specificity nucleic acid-binding domains, no significant affinity of the unusual PWI domain of Brr2 for a broad spectrum of DNAs and RNAs was detected in band-shift assays. Consistently, theC. thermophilumBrr2 PWI-like domain, in the conformation seen in the present crystal structure, lacks an expanded positively charged surface patch as observed in at least one canonical, nucleic acid-binding PWI domain. Instead, in a comprehensive yeast two-hybrid screen against human spliceosomal proteins, fragments of the N-terminal region of human Brr2 were found to interact with several other spliceosomal proteins. At least one of these interactions, with the Prp19 complex protein SPF27, depended on the presence of the PWI-like domain. The results suggest that the N-terminal region of Brr2 serves as a versatile protein–protein interaction platform in the spliceosome and that some interactions require or are reinforced by the PWI-like domain.

Crystal Structure and Solution NMR Studies of Lys48-linked Tetraubiquitin at Neutral pH

2007 ◽  
Vol 367 (1) ◽  
pp. 204-211 ◽  
Author(s):  
Michael J. Eddins ◽  
Ranjani Varadan ◽  
David Fushman ◽  
Cecile M. Pickart ◽  
Cynthia Wolberger
Keyword(s):  
Crystal Structure ◽  
Solution Nmr ◽  
Nmr Studies ◽  
Neutral Ph

Solution NMR studies of polytopic α-helical membrane proteins

2011 ◽  
Vol 21 (4) ◽  
pp. 497-508 ◽  
Author(s):  
Daniel Nietlispach ◽  
Antoine Gautier
Keyword(s):  
Membrane Proteins ◽  
Solution Nmr ◽  
Nmr Studies

scholarly journals Solution NMR studies of the integral membrane proteins OmpX and OmpA fromEscherichia coli

FEBS Letters ◽  
2001 ◽  
Vol 504 (3) ◽  
pp. 173-178 ◽  
Author(s):  
César Fernández ◽  
Christian Hilty ◽  
Sophie Bonjour ◽  
Koba Adeishvili ◽  
Konstantin Pervushin ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Integral Membrane Proteins ◽  
Solution Nmr ◽  
Nmr Studies ◽  
Fromescherichia Coli

scholarly journals ANTH domains within CALM, HIP1R, and Sla2 recognize ubiquitin internalization signals

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Natalya Pashkova ◽  
Lokesh Gakhar ◽  
Liping Yu ◽  
Nicholas J Schnicker ◽  
Annabel Y Minard ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Cell Surface ◽  
Terminal Region ◽  
Surface Proteins ◽  
Structural Studies ◽  
Cell Surface Proteins ◽  
Functional Studies ◽  
Binding Domains ◽  
Endocytic Machinery

Attachment of ubiquitin (Ub) to cell surface proteins serves as a signal for internalization via clathrin-mediated endocytosis (CME). How ubiquitinated membrane proteins engage the internalization apparatus remains unclear. The internalization apparatus contains proteins such as Epsin and Eps15, which bind Ub, potentially acting as adaptors for Ub-based internalization signals. Here we show that additional components of the endocytic machinery including CALM, HIP1R, and Sla2 bind Ub via their N-terminal ANTH domain, a domain belonging to the superfamily of ENTH and VHS domains. Structural studies revealed that Ub binds with µM affinity to a unique C-terminal region within the ANTH domain not found in ENTH domains. Functional studies showed that combined loss of Ub-binding by ANTH-domain proteins and other Ub-binding domains within the yeast internalization apparatus caused defects in the Ub-dependent internalization of the GPCR Ste2 that was engineered to rely exclusively on Ub as an internalization signal. In contrast, these mutations had no effect on the internalization of Ste2 engineered to use an alternate Ub-independent internalization signal. These studies define new components of the internalization machinery that work collectively with Epsin and Eps15 to specify recognition of Ub as an internalization signal.

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