Structure (v.19, #6)
In This Issue (v-vi).
Unveiled α−Neurexins Take Center Stage by Carsten Reissner; Markus Missler (749-750).
Neurexins are presynaptic transmembrane proteins that play an essential role in synapse function. The crystal structures of α−neurexin extracellular domains () provide important insights into their conformational freedom and their putative spatial arrangement with binding partners in the synaptic cleft.
Recombining DNA by Protein Swivels by Reid C. Johnson; Meghan M. McLean (751-753).
Two new reports on serine recombinases, one of a crystal snapshot in an alternate rotational conformer poised for DNA cleavage (), and a second employing single-DNA molecule approaches (), provide strong support for the subunit rotation model for exchanging DNA strands.
Structural Changes Underlying Allostery in Group II Chaperonins by Keith R. Willison (754-755).
The ATP-dependence of folding chamber closure in the 16-subunit homo-oligomeric chaperonin from archaea Methanococcus maripaludis (Mm-cpn) has been studied by single particle cryo-electron microscopy (). ATP binding alone causes a rigid body rotation of ∼45° and slight closure of the cavity, but full closure requires ATP hydrolysis.
Improved Technologies Now Routinely Provide Protein NMR Structures Useful for Molecular Replacement by Binchen Mao; Rongjin Guan; Gaetano T. Montelione (757-766).
Molecular replacement (MR) is widely used for addressing the phase problem in X-ray crystallography. Historically, crystallographers have had limited success using NMR structures as MR search models. Here, we report a comprehensive investigation of the utility of protein NMR ensembles as MR search models, using data for 25 pairs of X-ray and NMR structures solved and refined using modern NMR methods. Starting from NMR ensembles prepared by an improved protocol, FindCore, correct MR solutions were obtained for 22 targets. Based on these solutions, automatic model rebuilding could be done successfully. Rosetta refinement of NMR structures provided MR solutions for another two proteins. We also demonstrate that such properly prepared NMR ensembles and X-ray crystal structures have similar performance when used as MR search models for homologous structures, particularly for targets with sequence identity >40%.► Modern protein NMR structures are generally accurate enough for MR applications ► Variance matrix methods allow NMR structures to be used for MR applications ► Rosetta refinement can sometimes improve the phasing power of NMR structures
The Crystal Structure of the α-Neurexin-1 Extracellular Region Reveals a Hinge Point for Mediating Synaptic Adhesion and Function by Meghan T. Miller; Mauro Mileni; Davide Comoletti; Raymond C. Stevens; Michal Harel; Palmer Taylor (767-778).
α- and β-neurexins (NRXNs) are transmembrane cell adhesion proteins that localize to presynaptic membranes in neurons and interact with the postsynaptic neuroligins (NLGNs). Their gene mutations are associated with the autism spectrum disorders. The extracellular region of α-NRXNs, containing nine independently folded domains, has structural complexity and unique functional characteristics, distinguishing it from the smaller β-NRXNs. We have solved the X-ray crystal structure of seven contiguous domains of the α-NRXN-1 extracellular region at 3.0 Å resolution. The structure reveals an arrangement where the N-terminal five domains adopt a more rigid linear conformation and the two C-terminal domains form a separate arm connected by a flexible hinge. In an extended conformation the molecule is suitably configured to accommodate a bound NLGN molecule, as supported by structural comparison and surface plasmon resonance. These studies provide the structural basis for a multifunctional synaptic adhesion complex mediated by α-NRXN-1.► 3.0 Å structure of α-NRXN-1, LNS 2-6, reveals asymmetry and a flexible hinge ► Domain arrangement allows for simultaneous binding events ► A novel O-linked glycosylation site is revealed on the EGF2 domain ► α-NRXN binding to NLGN-1 has nM affinity and is governed by LNS 6
The Structure of Neurexin 1α Reveals Features Promoting a Role as Synaptic Organizer by Fang Chen; Vandavasi Venugopal; Beverly Murray; Gabby Rudenko (779-789).
α-Neurexins are essential synaptic adhesion molecules implicated in autism spectrum disorder and schizophrenia. The α-neurexin extracellular domain consists of six LNS domains interspersed by three EGF-like repeats and interacts with many different proteins in the synaptic cleft. To understand how α-neurexins might function as synaptic organizers, we solved the structure of the neurexin 1α extracellular domain (n1α) to 2.65 Å. The L-shaped molecule can be divided into a flexible repeat I (LNS1-EGF-A-LNS2), a rigid horseshoe-shaped repeat II (LNS3-EGF-B-LNS4) with structural similarity to so-called reelin repeats, and an extended repeat III (LNS5-EGF-B-LNS6) with controlled flexibility. A 2.95 Å structure of n1α carrying splice insert SS#3 in LNS4 reveals that SS#3 protrudes as a loop and does not alter the rigid arrangement of repeat II. The global architecture imposed by conserved structural features enables α-neurexins to recruit and organize proteins in distinct and variable ways, influenced by splicing, thereby promoting synaptic function.Display Omitted► The neurexin 1α extracellular domain (n1α) structure reveals an L-shaped molecule ► The three neurexin LNS-EGF-LNS repeats have different arrangements ► Repeat II, but not repeat I or III, shows structural similarity to reelin repeats ► The n1α architecture suggests how α-neurexins work as synaptic organizers
The Deletion of Exon 3 in the Cardiac Ryanodine Receptor Is Rescued by β Strand Switching by Paolo A. Lobo; Lynn Kimlicka; Ching-Chieh Tung; Filip Van Petegem (790-798).
Mutations in the cardiac Ryanodine Receptor (RYR2) are linked to triggered arrhythmias. Removal of exon 3 results in a severe form of catecholaminergic polymorphic ventricular tachycardia (CPVT). This exon encodes secondary structure elements that are crucial for folding of the N-terminal domain (NTD), raising the question of why the deletion is neither lethal nor confers a loss of function. We determined the 2.3 Å crystal structure of the NTD lacking exon 3. The removal causes a structural rescue whereby a flexible loop inserts itself into the β trefoil domain and increases thermal stability. The exon 3 deletion is not tolerated in the corresponding RYR1 domain. The rescue shows a novel mechanism by which RYR2 channels can adjust their Ca2+ release properties through altering the structure of the NTD. Despite the rescue, the deletion affects interfaces with other RYR2 domains. We propose that relative movement of the NTD is allosterically coupled to the pore region.► Deletion of RYR2 exon 3 does not result in a collapsed structure of the NTD ► A flexible loop rescues the beta trefoil core by becoming a β strand in RYR2 Δexon3 ► The rescue segment is unique to RYR2 and the exon 3 deletion is not tolerated in RYR1 ► Exon 3 deletion affects two major interfaces in intact RYR2
Structural Basis for Catalytic Activation of a Serine Recombinase by Ross A. Keenholtz; Sally-J. Rowland; Martin R. Boocock; W. Marshall Stark; Phoebe A. Rice (799-809).
Sin resolvase is a site-specific serine recombinase that is normally controlled by a complex regulatory mechanism. A single mutation, Q115R, allows the enzyme to bypass the entire regulatory apparatus, such that no accessory proteins or DNA sites are required. Here, we present a 1.86 Å crystal structure of the Sin Q115R catalytic domain, in a tetrameric arrangement stabilized by an interaction between Arg115 residues on neighboring subunits. The subunits have undergone significant conformational changes from the inactive dimeric state previously reported. The structure provides a new high-resolution view of a serine recombinase active site that is apparently fully assembled, suggesting roles for the conserved active site residues. The structure also suggests how the dimer-tetramer transition is coupled to assembly of the active site. The tetramer is captured in a different rotational substate than that seen in previous hyperactive serine recombinase structures, and unbroken crossover site DNA can be readily modeled into its active sites.Display Omitted► We report the 1.86 Å structure of an activated mutant of the Sin catalytic domain ► The active site appears to be fully assembled, primed for DNA cleavage ► Uncleaved DNA can be modeled into the active sites ► Observation of a new rotational substate supports the subunit rotation hypothesis
Domain Orientation in the N-Terminal PDZ Tandem from PSD-95 Is Maintained in the Full-Length Protein by James J. McCann; Liqiang Zheng; Salvatore Chiantia; Mark E. Bowen (810-820).
Tandem PDZ domains have been suggested to form structurally independent supramodules. However, dissimilarity between crystallography and NMR models emphasize their malleable conformation. Studies in full-length scaffold proteins are needed to examine the effect of tertiary interactions within their native context. Using single-molecule fluorescence to characterize the N-terminal PDZ tandem in PSD-95, we provide the first direct evidence that PDZ tandems can be structurally independent within a full-length scaffold protein. Molecular refinement using our data converged on a single structure with an antiparallel alignment of the ligand-binding sites. Devoid of interaction partners, single-molecule conditions captured PSD-95 in its unbound, ground state. Interactions between PDZ domains could not be detected while fluctuation correlation spectroscopy showed that other conformations are dynamically sampled. We conclude that ultra-weak interactions stabilize the conformation providing a “low-relief” energy landscape that allows the domain orientation to be flipped by environmental interactions.► Single-molecule FRET can reveal domain organization in full-length scaffold proteins ► The N−terminal PDZ tandem is structurally independent within full-length PSD-95 ► Ultra-weak interactions between PDZ domains result in a “low-relief” energy landscape ► The tandem has a lowest energy structure but dynamically samples other conformations
Implications for Damage Recognition during Dpo4-Mediated Mutagenic Bypass of m1G and m3C Lesions by Olga Rechkoblit; James C. Delaney; John M. Essigmann; Dinshaw J. Patel (821-832).
DNA is susceptible to alkylation damage by a number of environmental agents that modify the Watson-Crick edge of the bases. Such lesions, if not repaired, may be bypassed by Y-family DNA polymerases. The bypass polymerase Dpo4 is strongly inhibited by 1-methylguanine (m1G) and 3-methylcytosine (m3C), with nucleotide incorporation opposite these lesions being predominantly mutagenic. Further, extension after insertion of both correct and incorrect bases, introduces additional base substitution and deletion errors. Crystal structures of the Dpo4 ternary extension complexes with correct and mismatched 3′-terminal primer bases opposite the lesions reveal that both m1G and m3C remain positioned within the DNA template/primer helix. However, both correct and incorrect pairing partners exhibit pronounced primer terminal nucleotide distortion, being primarily evicted from the DNA helix when opposite m1G or misaligned when pairing with m3C. Our studies provide insights into mechanisms related to hindered and mutagenic bypass of methylated lesions and models associated with damage recognition by repair demethylases.Display Omitted► Dpo4 is strongly inhibited and highly mutagenic during bypass of m1G and m3C lesions ► In the Dpo4 extension complexes, m1G and m3C remain positioned within the DNA helix ► The 3′-terminal A, G, T, and C primer bases opposite the lesions are mispositioned ► Mechanisms of methylation damage recognition by repair demethylases are discussed
Hierarchical Binding of Cofactors to the AAA ATPase p97 by Petra Hänzelmann; Alexander Buchberger; Hermann Schindelin (833-843).
The hexameric AAA ATPase p97 is involved in several human proteinopathies and mediates ubiquitin-dependent protein degradation among other essential cellular processes. Via its N-terminal domain (N domain), p97 interacts with multiple regulatory cofactors including the UFD1/NPL4 heterodimer and members of the “ubiquitin regulatory X” (UBX) domain protein family; however, the principles governing cofactor selectivity remain to be deciphered. Our crystal structure of the FAS-associated factor 1 (FAF1)UBX domain in complex with the p97N domain reveals that the signature Phe-Pro-Arg motif known to be crucial for interactions of UBX domains with p97 adopts a cis-proline configuration, in contrast to a cis-trans mixture we derive for the isolated FAF1UBX domain. Biochemical studies confirm that binding critically depends on a proline at this position. Furthermore, we observe that the UBX proteins FAF1 and UBXD7 only bind to p97-UFD1/NPL4, but not free p97, thus demonstrating for the first time a hierarchy in p97-cofactor interactions.► FAF1UBX binds into the p97N domain hydrophobic interdomain CLEFT ► The conserved R…FPR signature of FAF1UBX features a cis-proline in the p97 complex ► A conserved binding mode for p97N adaptor proteins ► Hierarchical binding of cofactors to p97
A Smoothed Backbone-Dependent Rotamer Library for Proteins Derived from Adaptive Kernel Density Estimates and Regressions by Maxim V. Shapovalov; Roland L. Dunbrack (844-858).
Display Omitted► New backbone-dependent rotamer library ► Smoothed estimates of rotamer probabilities as function of backbone dihedrals ϕ,ψ ► Smoothed, accurate rotamer library for use in flexible-backbone structure prediction ► Full density estimates of nonrotameric dihedrals (e.g., χ2 of Asn) with ϕ,ψ
Relative Solvent Accessible Surface Area Predicts Protein Conformational Changes upon Binding by Joseph A. Marsh; Sarah A. Teichmann (859-867).
Protein interactions are often accompanied by significant changes in conformation. We have analyzed the relationships between protein structures and the conformational changes they undergo upon binding. Based upon this, we introduce a simple measure, the relative solvent accessible surface area, which can be used to predict the magnitude of binding-induced conformational changes from the structures of either monomeric proteins or bound subunits. Applying this to a large set of protein complexes suggests that large conformational changes upon binding are common. In addition, we observe considerable enrichment of intrinsically disordered sequences in proteins predicted to undergo large conformational changes. Finally, we demonstrate that the relative solvent accessible surface area of monomeric proteins can be used as a simple proxy for protein flexibility. This reveals a powerful connection between the flexibility of unbound proteins and their binding-induced conformational changes, consistent with the conformational selection model of molecular recognition.Display Omitted► Relative solvent accessible surface area (A rel) is simple to calculate ► A rel predicts conformational changes upon binding from monomers or bound subunits ► Analysis of protein complexes suggests large conformational changes are common ► Intrinsic protein flexibility correlates with conformational changes upon binding
The Cap-Binding Translation Initiation Factor, eIF4E, Binds a Pseudoknot in a Viral Cap-Independent Translation Element by Zhaohui Wang; Marc Parisien; Kay Scheets; W. Allen Miller (868-880).
Eukaryotic initiation factor eIF4E performs a key early step in translation by specifically recognizing the m7GpppN cap structure at the 5′ end of cellular mRNAs. Many viral mRNAs lack a 5′ cap and thus bypass eIF4E. In contrast, we reported a cap-independent translation element (PTE) in Pea enation mosaic virus RNA2 that binds and requires eIF4E for translation initiation. To understand how this uncapped RNA is bound tightly by eIF4E, we employ SHAPE probing, phylogenetic comparisons with new PTEs discovered in panico- and carmoviruses, footprinting of the eIF4E binding site, and 3D RNA modeling using NAST, MC-Fold, and MC-Sym to predict a compact, 3D structure of the RNA. We propose that the cap-binding pocket of eIF4E clamps around a pseudoknot, placing a highly SHAPE-reactive guanosine in the pocket in place of the normal m7GpppN cap. This reveals a new mechanism of mRNA recognition by eIF4E.Display Omitted► Several plant viruses have a 3′ cap-independent translation element that binds eIF4E ► SHAPE probing reveals a compact pseudoknot with a hypermodified guanosine ► 3D RNA modeling suggests how an uncapped RNA may bind eIF4E at the cap-binding pocket ► This is a new mechanism by which a viral 3′ UTR element captures the eIF4F complex
A Systematic Study of the Energetics Involved in Structural Changes upon Association and Connectivity in Protein Interaction Networks by Amelie Stein; Manuel Rueda; Alejandro Panjkovich; Modesto Orozco; Patrick Aloy (881-889).
The study of protein binding mechanisms is a major topic of research in structural biology. Here, we implement a combination of metrics to systematically assess the cost of backbone conformational changes that protein domains undergo upon association. Through the analyses of 2090 unique unbound → bound transitions, from over 12,000 structures, we show that two-thirds of these proteins do not suffer significant structural changes upon binding, and could thus fit the lock-and-key model well. Among the remaining proteins, one-third explores the bound conformation in the unbound state (conformational selection model) and, while most transitions are possible from an energetic perspective, a few do require external help to break the thermodynamic barrier (induced fit model). We also analyze the relationship between conformational transitions and protein connectivity, finding that, in general, domains interacting with many partners undergo smaller changes upon association, and are less likely to freely explore larger conformational changes.Display Omitted► 65% of the proteins tested do not undergo major conformational changes upon binding ► 13% explore their bound conformations in their unbound state ► Only 2% require a external energy push to reach their bound conformation ► Hub proteins do not freely explore their bound conformations while unbound
Fine Details of IGF-1R Activation, Inhibition, and Asymmetry Determined by Associated Hydrogen /Deuterium-Exchange and Peptide Mass Mapping by Damian Houde; Stephen J. Demarest (890-900).
The structural features of the asymmetric activated states of the insulin receptor family are still poorly understood. We investigated hydrogen/deuterium (H/D)-exchange within the extracellular domain of the type-I insulin-like growth factor receptor (IGF-1R) in the absence and presence of IGF-1 (active state) and in the presence of antibody inhibitors (inactive state). Near complete coverage of the 210 kDa receptor sequence was obtained by mass mapping of proteolytically derived peptides at all H/D-exchange time points. The data provide details regarding solvent exposure and dynamics across the extracellular region as well as conformational changes induced by activation or inactivation. Multiple peptides, distant in structure, individually demonstrated two distinct H/D-exchange rates, suggesting that each of these peptides exists in two separate environments in IGF-1R. The dual-exchange behavior of these peptides was enhanced on ligand binding and eliminated on inhibitor binding, clearly associating these regions with active state asymmetry and enabling them to serve as reporters of receptor activity.► Mapping of regions that exist in asymmetric environments within an IGF-1R homodimer ► Association of asymmetric populations with the activated state of the receptor ► H/D-exchange dynamics across the entire IGF-1R extracellular domain (210 kDa) ► Changes in H/D-exchange within IGF-1R resulting from ligand or antagonist binding