Structure (v.19, #11)
In This Issue (v-vii).
Cutting-Hedge Research into Bacterial Invasion by Douglas A. Kuntz; David R. Rose (1535-1536).
Glycoside hydrolases are the tools that pathogenic bacteria use to cut through the defensive glycan structure on host cell surfaces. In this issue of Structure, Pluvinage et al. (2011) report how a bacterial polypeptide with more than one hydrolase module broadens the effective substrate specificity.
Fibrinogen Unfolding Mechanisms Are Not Too Much of a Stretch by Martin Guthold; Samuel S. Cho (1536-1538).
Molecular explanations for the extraordinary elasticity and extensibility of fibrin fibers are still lacking. Now, Zhmurov et al. (2011) use force spectroscopy experiments, and innovative simulations that match the time and force scales of these experiments, to study fibrinogen behavior under an applied force providing deeper insights into this process.
Tackling the Legs of Mannan-Binding Lectin by Erhard Hohenester (1538-1540).
The recognition of pathogen surfaces by mannan-binding lectin activates MASP proteases, leading to complement activation. A crystal structure by Gingras et al. (2011) in this issue of Structure now shows how the collagen-like stems of mannan-binding lectin bind MASP-1 through a minimalist set of interactions.
Type II ABC Permeases: Are They Really So Different? by Anthony M. George; Peter M. Jones (1540-1542).
Structural and biochemical data reported by Tirado-Lee et al. (2011) in this issue of Structure reveal the existence of high and low affinity ABC transporters for the same substrate in a single organism, thus raising questions about structural and mechanistic differences within the ABC superfamily.
Cell Membranes: The Lipid Perspective by Ünal Coskun; Kai Simons (1543-1548).
Although cell membranes are packed with proteins mingling with lipids, remarkably little is known about how proteins interact with lipids to carry out their function. Novel analytical tools are revealing the astounding diversity of lipids in membranes. The issue is now to understand the cellular functions of this complexity. In this Perspective, we focus on the interface of integral transmembrane proteins and membrane lipids in eukaryotic cells. Clarifying how proteins and lipids interact with each other will be important for unraveling membrane protein structure and function. Progress toward this goal will be promoted by increasing overlap between different fields that have so far operated without much crosstalk.
Toward the Fourth Dimension of Membrane Protein Structure: Insight into Dynamics from Spin-Labeling EPR Spectroscopy by Hassane S. Mchaourab; P. Ryan Steed; Kelli Kazmier (1549-1561).
Trapping membrane proteins in the confines of a crystal lattice obscures dynamic modes essential for interconversion between multiple conformations in the functional cycle. Moreover, lattice forces could conspire with detergent solubilization to stabilize a minor conformer in an ensemble thus confounding mechanistic interpretation. Spin labeling in conjunction with electron paramagnetic resonance (EPR) spectroscopy offers an exquisite window into membrane protein dynamics in the native-like environment of a lipid bilayer. Systematic application of spin labeling and EPR identifies sequence-specific secondary structures, defines their topology and their packing in the tertiary fold. Long range distance measurements (60 Å–80 Å) between pairs of spin labels enable quantitative analysis of equilibrium dynamics and triggered conformational changes. This review highlights the contribution of spin labeling to bridging structure and mechanism. Efforts to develop methods for determining structures from EPR restraints and to increase sensitivity and throughput promise to expand spin labeling applications in membrane protein structural biology.
Molecular Simulation Approaches to Membrane Proteins by Phillip J. Stansfeld; Mark S.P. Sansom (1562-1572).
Molecular simulations are an invaluable tool for understanding membrane proteins. Improvements to both hardware and simulation methods have allowed access to physiologically relevant timescales and have permitted the simulation of large multimeric complexes. This, coupled to the recent expansion in membrane protein structures, provides a means to elucidate the relationship between protein structure and function. In this review, we discuss the progress in using simulations to understand the complex processes that occur at the boundary of a cell, ranging from the transport of solutes and the interactions of ligands with ion channels to the conformational rearrangements required for gating of channels and the signaling by membrane-associated complexes.
Direct Visualization of HIV-1 with Correlative Live-Cell Microscopy and Cryo-Electron Tomography by Sangmi Jun; Danxia Ke; Karl Debiec; Gongpu Zhao; Xin Meng; Zandrea Ambrose; Gregory A. Gibson; Simon C. Watkins; Peijun Zhang (1573-1581).
Cryo-electron tomography (cryoET) allows 3D visualization of cellular structures at molecular resolution in a close-to-native state and therefore has the potential to help elucidate early events of HIV-1 infection in host cells. However, structural details of infecting HIV-1 have not been observed, due to technological challenges in working with rare and dynamic HIV-1 particles in human cells. Here, we report structural analysis of HIV-1 and host-cell interactions by means of a correlative high-speed 3D live-cell-imaging and cryoET method. Using this method, we showed under near-native conditions that intact hyperstable mutant HIV-1 cores are released into the cytoplasm of host cells. We further obtained direct evidence to suggest that a hyperstable mutant capsid, E45A, showed delayed capsid disassembly compared to the wild-type capsid. Together, these results demonstrate the advantages of our correlative live-cell and cryoET approach for imaging dynamic processes, such as viral infection.Display Omitted► We developed a correlative 3D live-cell and cryoET approach to image HIV-1 infection ► We built an integrated cryostage for both cryofluorescence microscopy and cryoEM ► Intact HIV-1 cores were directly visualized in the cytoplasm of host cells ► HIV-1 E45A cores undergo delayed capsid disassembly in target cells
Identifying Conformational States of Macromolecules by Eigen-Analysis of Resampled Cryo-EM Images by Pawel A. Penczek; Marek Kimmel; Christian M.T. Spahn (1582-1590).
We present the codimensional principal component analysis (PCA), a novel and straightforward method for resolving sample heterogeneity within a set of cryo-EM 2D projection images of macromolecular assemblies. The method employs PCA of resampled 3D structures computed using subsets of 2D data obtained with a novel hypergeometric sampling scheme. PCA provides us with a small subset of dominating “eigenvolumes” of the system, whose reprojections are compared with experimental projection data to yield their factorial coordinates constructed in a common framework of the 3D space of the macromolecule. Codimensional PCA is unique in the dramatic reduction of dimensionality of the problem, which facilitates rapid determination of both the plausible number of conformers in the sample and their 3D structures. We applied the codimensional PCA to a complex data set of Thermus thermophilus 70S ribosome, and we identified four major conformational states and visualized high mobility of the stalk base region.► Codimensional PCA was developed for analysis of EM sample heterogeneity ► The method is based on hypergeometric stratified resampling of 2D EM data ► It yields correct eigenvectors of 3D data set as represented by its 2D projections ► Codimensional PCA correctly identifies conformers in the EM sample
Crystal Structure of Human Mre11: Understanding Tumorigenic Mutations by Young Bong Park; Jina Chae; Young Chang Kim; Yunje Cho (1591-1602).
Mre11 plays an important role in repairing damaged DNA by cleaving broken ends and by providing a platform for other DNA repair proteins. Various Mre11 mutations have been identified in several types of cancer. We have determined the crystal structure of the human Mre11 core (hMre11), which contains the nuclease and capping domains. hMre11 dimerizes through the interfaces between loop β3-α3 from one Mre11 and loop β4-β5 from another Mre11, and between loop α2-β3 from one Mre11 and helices α2 and α3 from another Mre11, and assembles into a completely different dimeric architecture compared with bacterial or archaeal Mre11 homologs. Nbs1 binds to the region containing loop α2-β3 which participates in dimerization. The hMre11 structure in conjunction with biochemical analyses reveals that many tumorigenic mutations are primarily associated with Nbs1 binding and partly with nuclease activities, providing a framework for understanding how mutations inactivate Mre11.Display Omitted► We have determined the crystal structure of the human Mre11 core ► Human Mre11 assembles into a novel dimeric architecture ► Nbs1 binds to the region containing loop α2-β3 which participates in dimerization ► Many tumorigenic mutations are primarily associated with Nbs1 binding and nuclease activities
Inhibition of the Pneumococcal Virulence Factor StrH and Molecular Insights into N-Glycan Recognition and Hydrolysis by Benjamin Pluvinage; Melanie A. Higgins; D. Wade Abbott; Craig Robb; Ankur B. Dalia; Lehua Deng; Jeffrey N. Weiser; Thomas B. Parsons; Antony J. Fairbanks; David J. Vocadlo; Alisdair B. Boraston (1603-1614).
The complete degradation of N-linked glycans by the pathogenic bacterium Streptococcus pneumoniae is facilitated by the large multimodular cell wall-attached exo-β-D-N-acetylglucosaminidase StrH. Structural dissection of this virulence factor using X-ray crystallography showed it to have two structurally related glycoside hydrolase family 20 catalytic domains, which displayed the expected specificity for complex N-glycans terminating in N-acetylglucosamine but exhibited unexpected differences in their preferences for the substructures present in these glycans. The structures of the two catalytic domains in complex with unhydrolyzed substrates, including an N-glycan possessing a bisecting N-acetylglucosamine residue, revealed the specific architectural features in the active sites that confer their differential specificities. Inhibitors of StrH are demonstrated to be effective tools in modulating the interaction of StrH with components of the host, such as the innate immune system. Overall, new structural and functional insight into a carbohydrate-mediated component of the pneumococcus-host interaction is provided.► The catalytic modules of StrH possess subtly different specificities ► The structural basis of StrH's specificity for glycans ► The structure of a glycoside hydrolase in complex with a bisected N-glycan ► Modulation of StrH's biological roles using inhibitors.
Mechanism of Fibrin(ogen) Forced Unfolding by Artem Zhmurov; Andre E.X. Brown; Rustem I. Litvinov; Ruxandra I. Dima; John W. Weisel; Valeri Barsegov (1615-1624).
Fibrinogen, upon enzymatic conversion to monomeric fibrin, provides the building blocks for fibrin polymer, the scaffold of blood clots and thrombi. Little has been known about the force-induced unfolding of fibrin(ogen), even though it is the foundation for the mechanical and rheological properties of fibrin, which are essential for hemostasis. We determined mechanisms and mapped the free energy landscape of the elongation of fibrin(ogen) monomers and oligomers through combined experimental and theoretical studies of the nanomechanical properties of fibrin(ogen), using atomic force microscopy-based single-molecule unfolding and simulations in the experimentally relevant timescale. We have found that mechanical unraveling of fibrin(ogen) is determined by the combined molecular transitions that couple stepwise unfolding of the γ chain nodules and reversible extension-contraction of the α-helical coiled-coil connectors. These findings provide important characteristics of the fibrin(ogen) nanomechanics necessary to understand the molecular origins of fibrin viscoelasticity at the fiber and whole clot levels.► Forced elongation of fibrin(ogen) is determined mainly by unfolding of the γ nodules ► C-term β strand insert supports the integral multidomain structure of the γ nodule ► The α-helical coil-coiled connectors act as highly elastic molecular capacitors ► The mechanical unfolding of the full length Fg is mostly an enthalpy driven process
Structural Basis for the Recognition of Phosphorylated Histone H3 by the Survivin Subunit of the Chromosomal Passenger Complex by A. Arockia Jeyaprakash; Claire Basquin; Uma Jayachandran; Elena Conti (1625-1634).
Localization of the chromosomal passenger complex (CPC) at centromeres during early mitosis is essential for accurate chromosome segregation and is dependent on the phosphorylation of histone H3. We report the 2.7 Å resolution structure of the CPC subunit Survivin bound to the N-terminal tail of histone H3 carrying the Thr3 phosphorylation mark (Thr3ph). The BIR domain of Survivin recognizes the Ala1-Arg2-Thr3ph-Lys4 sequence, decoding the modification state and the free N terminus of histone H3 by a strategy similar to that used by PHD fingers. The structural analysis permitted the identification of putative Survivin-binding epitopes in other mitotic proteins, including human Shugoshin 1. Using biophysical and structural data, we show that a phospho-mimic N-terminal sequence such as that of hSgo1 (Ala1-Lys2-Glu3-Arg4) contains the specificity determinants to bind Survivin. Our findings suggest that the CPC engages in mutually exclusive interactions with other constituents of the mitotic machinery and a histone mark in chromatin.► The BIR domain of Survivin specifically recognizes Ala1-Arg2-Thr3ph-Lys4 of histone H3 ► BIR and PHD finger domains employ a common mode of N-terminal peptide recognition ► Survivin-binding epitopes are present in several mitotic proteins ► Survivin directly binds to Ala1-Lys2-Glu3-Arg4 of Shugoshin 1 in vitro
Structural Basis of Mannan-Binding Lectin Recognition by Its Associated Serine Protease MASP-1: Implications for Complement Activation by Alexandre R. Gingras; Umakhanth Venkatraman Girija; Anthony H. Keeble; Roshni Panchal; Daniel A. Mitchell; Peter C.E. Moody; Russell Wallis (1635-1643).
Complement activation contributes directly to health and disease. It neutralizes pathogens and stimulates immune processes. Defects lead to immunodeficiency and autoimmune diseases, whereas inappropriate activation causes self-damage. In the lectin and classical pathways, complement is triggered upon recognition of a pathogen by an activating complex. Here we present the first structure of such a complex in the form of the collagen-like domain of mannan-binding lectin (MBL) and the binding domain of its associated protease (MASP-1/-3). The collagen binds within a groove using a pivotal lysine side chain that interacts with Ca2+-coordinating residues, revealing the essential role of Ca2+. This mode of binding is prototypic for all activating complexes of the lectin and classical pathways, and suggests a general mechanism for the global changes that drive activation. The structural insights reveal a new focus for inhibitors and we have validated this concept by targeting the binding pocket of the MASP.Display Omitted► Structure of an activating complex of complement at 1.8 Å resolution ► Complex reveals a novel mechanism for collagen recognition and highlights a conserved mode of binding via Ca2+-dependent CUB domains ► Multiple weak interactions between components mediate the global changes that initiate complement activation ► Binding interface is a viable target for small molecule inhibitors of complement
Enhanced Selectivity for Sulfatide by Engineered Human Glycolipid Transfer Protein by Valeria R. Samygina; Alexander N. Popov; Aintzane Cabo-Bilbao; Borja Ochoa-Lizarralde; Felipe Goni-de-Cerio; Xiuhong Zhai; Julian G. Molotkovsky; Dinshaw J. Patel; Rhoderick E. Brown; Lucy Malinina (1644-1654).
Human glycolipid transfer protein (GLTP) fold represents a novel structural motif for lipid binding/transfer and reversible membrane translocation. GLTPs transfer glycosphingolipids (GSLs) that are key regulators of cell growth, division, surface adhesion, and neurodevelopment. Herein, we report structure-guided engineering of the lipid binding features of GLTP. New crystal structures of wild-type GLTP and two mutants (D48V and A47D‖D48V), each containing bound N-nervonoyl-sulfatide, reveal the molecular basis for selective anchoring of sulfatide (3-O-sulfo-galactosylceramide) by D48V-GLTP. Directed point mutations of “portal entrance” residues, A47 and D48, reversibly regulate sphingosine access to the hydrophobic pocket via a mechanism that could involve homodimerization. “Door-opening” conformational changes by phenylalanines within the hydrophobic pocket are revealed during lipid encapsulation by new crystal structures of bona fide apo-GLTP and GLTP complexed with N-oleoyl-glucosylceramide. The development of “engineered GLTPs” with enhanced specificity for select GSLs provides a potential new therapeutic approach for targeting GSL-mediated pathologies.Display Omitted► D48V mutation drastically enhances transfer selectivity of human GLTP for sulfatide ► D48V mutation switches the lipid chain binding mode within GLTP ► A47D mutation added to D48V-GLTP restores the lipid chain binding mode
Structural Investigation of Rimantadine Inhibition of the AM2-BM2 Chimera Channel of Influenza Viruses by Rafal M. Pielak; Kirill Oxenoid; James J. Chou (1655-1663).
The M2 channel of influenza A is a target of the adamantane family antiviral drugs. Two different drug-binding sites have been reported: one inside the pore, and the other is a lipid-facing pocket. A previous study showed that a chimera of M2 variants from influenza A and B that contains only the pore-binding site is sensitive to amantadine inhibition, suggesting that the primary site of inhibition is inside the pore. To obtain atomic details of channel-drug interaction, we determined the structures of the chimeric channel with and without rimantadine. Inside the channel and near the N-terminal end, methyl groups of Val27 and Ala30 from four subunits form a hydrophobic pocket around the adamantane, and the drug amino group appears to be in polar contact with the backbone oxygen of Ala30. The structures also reveal differences between the drug-bound and -unbound states of the channel that can explain drug resistance.Display Omitted► The AM2-BM2 channel is relevant for studying drug binding of influenza M2 channel ► Obtained detailed structures of the chimeric channel with and without rimantadine ► Rimantadine binding significantly alters the structure of the channel pore ► The NMR system of the AM2-BM2 channel is useful for investigating drug inhibition
Membrane Binding of the N-Terminal Ubiquitin-Like Domain of kindlin-2 Is Crucial for Its Regulation of Integrin Activation by H. Dhanuja Perera; Yan-Qing Ma; Jun Yang; Jamila Hirbawi; Edward F. Plow; Jun Qin (1664-1671).
Kindlin-2 belongs to an emerging class of regulators for heterodimeric (α/β) integrin adhesion receptors. By binding to integrin β cytoplasmic tail via its C-terminal FERM-like domain, kindlin-2 promotes integrin activation. Intriguingly, this activation process depends on the N terminus of kindlin-2 (K2-N) that precedes the FERM domain. The molecular function of K2-N is unclear. We present the solution structure of K2-N, which displays a ubiquitin fold similar to that observed in kindlin-1. Using chemical shift mapping and mutagenesis, we found that K2-N contains a conserved positively charged surface that binds to membrane enriched with negatively charged phosphatidylinositol-(4,5)-bisphosphate. We show that while wild-type kindlin-2 is capable of promoting integrin activation, such ability is significantly reduced for its membrane-binding defective mutant. These data suggest a membrane-binding function of the ubiquitin-like domain of kindlin-2, which is likely common for all kindlins to promote their localization to the plasma membrane and control integrin activation.► Kindlin-2 adaptor has a FERM domain that binds and activates integrin ► A ubiquitin-like N terminus preceding the FERM domain controls the kindlin-2 function ► The kindlin-2 ubiquitin domain binds to the negatively charged membrane surface ► The membrane binding to the kindlin-2 ubiquitin domain promotes integrin activation
Structural Insights into Ail-Mediated Adhesion in Yersinia pestis by Satoshi Yamashita; Petra Lukacik; Travis J. Barnard; Nicholas Noinaj; Suleyman Felek; Tiffany M. Tsang; Eric S. Krukonis; B. Joseph Hinnebusch; Susan K. Buchanan (1672-1682).
Ail is an outer membrane protein from Yersinia pestis that is highly expressed in a rodent model of bubonic plague, making it a good candidate for vaccine development. Ail is important for attaching to host cells and evading host immune responses, facilitating rapid progression of a plague infection. Binding to host cells is important for injection of cytotoxic Yersinia outer proteins. To learn more about how Ail mediates adhesion, we solved two high-resolution crystal structures of Ail, with no ligand bound and in complex with a heparin analog called sucrose octasulfate. We identified multiple adhesion targets, including laminin and heparin, and showed that a 40 kDa domain of laminin called LG4-5 specifically binds to Ail. We also evaluated the contribution of laminin to delivery of Yops to HEp-2 cells. This work constitutes a structural description of how a bacterial outer membrane protein uses a multivalent approach to bind host cells.Display Omitted► Ail is an 8-stranded β-barrel having 4 extracellular loops that function in adhesion ► Ail-laminin and Ail-fibronectin interactions mediate delivery of Yops to host cells ► Ail has two binding sites for heparin that also confer adhesion to host cells
Structural Basis for μ-Opioid Receptor Binding and Activation by Adrian W.R. Serohijos; Shuangye Yin; Feng Ding; Josee Gauthier; Dustin G. Gibson; William Maixner; Nikolay V. Dokholyan; Luda Diatchenko (1683-1690).
Opioids that stimulate the μ-opioid receptor (MOR1) are the most frequently prescribed and effective analgesics. Here we present a structural model of MOR1. Molecular dynamics simulations show a ligand-dependent increase in the conformational flexibility of the third intracellular loop that couples with the G protein complex. These simulations likewise identified residues that form frequent contacts with ligands. We validated the binding residues using site-directed mutagenesis coupled with radioligand binding and functional assays. The model was used to blindly screen a library of ∼1.2 million compounds. From the 34 compounds predicted to be strong binders, the top three candidates were examined using biochemical assays. One compound showed high efficacy and potency. Post hoc testing revealed this compound to be nalmefene, a potent clinically used antagonist, thus further validating the model. In summary, the MOR1 model provides a tool for elucidating the structural mechanism of ligand-initiated cell signaling and for screening novel analgesics.► A novel μ-opioid receptor (MOR1) in silico model was build and validated by site-directed mutagenesis studies ► Molecular dynamics simulations show a ligand-dependent increase in the conformational flexibility of the third intracellular loop of the model receptor ► Blind in silico screening of the library of ∼1.2 million compounds recapitulates a known clinically used antagonist
Structure of a Blinkin-BUBR1 Complex Reveals an Interaction Crucial for Kinetochore-Mitotic Checkpoint Regulation via an Unanticipated Binding Site by Victor M. Bolanos-Garcia; Tiziana Lischetti; Dijana Matak-Vinković; Ernesto Cota; Pete J. Simpson; Dimitri Y. Chirgadze; David R. Spring; Carol V. Robinson; Jakob Nilsson; Tom L. Blundell (1691-1700).
The maintenance of genomic stability relies on the spindle assembly checkpoint (SAC), which ensures accurate chromosome segregation by delaying the onset of anaphase until all chromosomes are properly bioriented and attached to the mitotic spindle. BUB1 and BUBR1 kinases are central for this process and by interacting with Blinkin, link the SAC with the kinetochore, the macromolecular assembly that connects microtubules with centromeric DNA. Here, we identify the Blinkin motif critical for interaction with BUBR1, define the stoichiometry and affinity of the interaction, and present a 2.2 Å resolution crystal structure of the complex. The structure defines an unanticipated BUBR1 region responsible for the interaction and reveals a novel Blinkin motif that undergoes a disorder-to-order transition upon ligand binding. We also show that substitution of several BUBR1 residues engaged in binding Blinkin leads to defects in the SAC, thus providing the first molecular details of the recognition mechanism underlying kinetochore-SAC signaling.Display Omitted► Molecular details of the recognition mechanism underlying kinetochore-SAC signalling ► Crystal structure of a BUBR1-Blinkin complex defines an unanticipated BUBR1 region ► A novel Blinkin motif undergoes a disorder-to-order transition upon BUBR1 binding ► Mutation of BUBR1 residues that bind Blinkin lead to an impaired mitotic checkpoint
Classification of a Haemophilus influenzae ABC Transporter HI1470/71 through Its Cognate Molybdate Periplasmic Binding Protein, MolA by Leidamarie Tirado-Lee; Allen Lee; Douglas C. Rees; Heather W. Pinkett (1701-1710).
molA (HI1472) from H. influenzae encodes a periplasmic binding protein (PBP) that delivers substrate to the ABC transporter MolB2C2 (formerly HI1470/71). The structures of MolA with molybdate and tungstate in the binding pocket were solved to 1.6 and 1.7 Å resolution, respectively. The MolA-binding protein binds molybdate and tungstate, but not other oxyanions such as sulfate and phosphate, making it the first class III molybdate-binding protein structurally solved. The ∼100 μM binding affinity for tungstate and molybdate is significantly lower than observed for the class II ModA molybdate-binding proteins that have nanomolar to low micromolar affinity for molybdate. The presence of two molybdate loci in H. influenzae suggests multiple transport systems for one substrate, with molABC constituting a low-affinity molybdate locus.► MolA is a class III molybdate periplasmic binding protein (PBP), which delivers substrate to MolB2C2, the ABC transporter from H. influenzae ► Structures of MolA were solved to 1.6 and 1.7 Å resolutions bound to molybdate and tungstate, respectively ► MolA is distinguished from the more common class II ModA PBP by the presence of a hinge helix connecting two globular domains and a significantly lower binding affinity for molybdate ► The presence of two molybdate loci indicates multiple molybdate transport systems for low and high-affinity transport are present in H. influenzae
A Conformational Switch in the CRIB-PDZ Module of Par-6 by Dustin S. Whitney; Francis C. Peterson; Brian F. Volkman (1711-1722).
Here, we report a novel mechanism of PDZ (PSD-95/Dlg/ZO-1) domain regulation that distorts a conserved element of PDZ ligand recognition. The polarity regulator Par-6 assembles a conserved multiprotein complex and is directly modulated by the Rho GTPase Cdc42. Cdc42 binds the adjacent Cdc42/Rac interactive binding (CRIB) and PDZ domains of Par-6, increasing C-terminal ligand binding affinity by 10-fold. By solving structures of the isolated PDZ domain and a disulfide-stabilized CRIB-PDZ, we detected a conformational switch that controls affinity by altering the configuration of the conserved “GLGF” loop. As a result, lysine 165 is displaced from the PDZ core by an adjacent hydrophobic residue, disrupting coordination of the PDZ ligand-binding cleft. Stabilization of the CRIB:PDZ interface restores K165 to its canonical location in the binding pocket. We conclude that a unique “dipeptide switch” in the Par-6 PDZ transmits a signal for allosteric activation to the ligand-binding pocket.► Par-6 CRIB-PDZ module is regulated by interdomain allostery ► Par-6 PDZ exists in a equilibrium between low- and high-affinity states ► Displacement of a conserved lysine creates a conformational PDZ switch ► Dipeptide switch in Par-6 is unprecedented in the PDZ family