Clathrin Lattice Research Articles

A balance between membrane elasticity and polymerization energy sets the shape of spherical clathrin coats.

In endocytosis, scaffolding is one of the mechanisms to create membrane curvature by moulding the membrane into the spherical shape of the clathrin cage. However, the impact of membrane elastic parameters on the assembly and shape of clathrin lattices has never been experimentally evaluated. Here, we show that membrane tension opposes clathrin polymerization. We reconstitute clathrin budding in vitro with giant unilamellar vesicles (GUVs), purified adaptors and clathrin. By changing the osmotic conditions, we find that clathrin coats cause extensive budding of GUVs under low membrane tension while polymerizing into shallow pits under moderate tension. High tension fully inhibits polymerization. Theoretically, we predict the tension values for which transitions between different clathrin coat shapes occur. We measure the changes in membrane tension during clathrin polymerization, and use our theoretical framework to estimate the polymerization energy from these data. Our results show that membrane tension controls clathrin-mediated budding by varying the membrane budding energy.

Membrane indentation triggers clathrin lattice reorganization and fluidization.

Clathrin-mediated endocytosis involves the coordinated assembly of clathrin cages around membrane indentations, necessitating fluid-like reorganization followed by solid-like stabilization. This apparent duality in clathrin's in vivo behavior provides some indication that the physical interactions between clathrin triskelia and the membrane effect a local response that triggers fluid-solid transformations within the clathrin lattice. We develop a computational model to study the response of clathrin protein lattices to spherical deformations of the underlying flexible membrane. These deformations are similar to the shapes assumed during intracellular trafficking of nanoparticles. Through Monte Carlo simulations of clathrin-on-membrane systems, we observe that these membrane indentations give rise to a greater than normal defect density within the overlaid clathrin lattice. In many cases, the bulk surrounding lattice remains in a crystalline phase, and the extra defects are localized to the regions of large curvature. This can be explained by the fact that the in-plane elastic stress in the clathrin lattice are reduced by coupling defects to highly curved regions. The presence of defects brought about by indentation can result in the fluidization of a lattice that would otherwise be crystalline, resulting in an indentation-driven, defect-mediated phase transition. Altering subunit elasticity or membrane properties is shown to drive a similar transition, and we present phase diagrams that map out the combined effects of these parameters on clathrin lattice properties.

Flat clathrin lattices: stable features of the plasma membrane.

Clathrin-mediated endocytosis (CME) is a fundamental property of eukaryotic cells. Classical CME proceeds via the formation of clathrin-coated pits (CCPs) at the plasma membrane, which invaginate to form clathrin-coated vesicles, a process that is well understood. However, clathrin also assembles into flat clathrin lattices (FCLs); these structures remain poorly described, and their contribution to cell biology is unclear. We used quantitative imaging to provide the first comprehensive description of FCLs and explore their influence on plasma membrane organization. Ultrastructural analysis by electron and superresolution microscopy revealed two discrete populations of clathrin structures. CCPs were typified by their sphericity, small size, and homogeneity. FCLs were planar, large, and heterogeneous and present on both the dorsal and ventral surfaces of cells. Live microscopy demonstrated that CCPs are short lived and culminate in a peak of dynamin recruitment, consistent with classical CME. In contrast, FCLs were long lived, with sustained association with dynamin. We investigated the biological relevance of FCLs using the chemokine receptor CCR5 as a model system. Agonist activation leads to sustained recruitment of CCR5 to FCLs. Quantitative molecular imaging indicated that FCLs partitioned receptors at the cell surface. Our observations suggest that FCLs provide stable platforms for the recruitment of endocytic cargo.

Membrane Fluctuations Destabilize Clathrin Protein Lattice Order

We develop a theoretical model of a clathrin protein lattice on a flexible cell membrane. The clathrin subunit is modeled as a three-legged pinwheel with elastic deformation modes and intersubunit binding interactions. The pinwheels are constrained to lie on the surface of an elastic sheet that opposes bending deformation and is subjected to tension. Through Monte Carlo simulations, we predict the equilibrium phase behavior of clathrin lattices at various levels of tension. High membrane tensions, which correspond to suppressed membrane fluctuations, tend to stabilize large, flat crystalline structures similar to plaques that have been observed in vivo on cell membranes that are adhered to rigid surfaces. Low tensions, on the other hand, give rise to disordered, defect-ridden lattices that behave in a fluidlike manner. The principles of two-dimensional melting theory are applied to our model system to further clarify how high tensions can stabilize crystalline order on flexible membranes. These results demonstrate the importance of environmental physical cues in dictating the collective behavior of self-assembled protein structures.

Regulation of Endocytic Clathrin Dynamics by Cargo Ubiquitination

SummarySome endocytic cargoes control clathrin-coated pit (CCP) maturation, but it is not known how such regulation is communicated. We found that μ-opioid neuropeptide receptors signal to their enclosing CCPs by ubiquitination. Nonubiquitinated receptors delay CCPs at an intermediate stage of maturation, after clathrin lattice assembly is complete but before membrane scission. Receptor ubiquitination relieves this inhibition, effectively triggering CCP scission and producing a receptor-containing endocytic vesicle. The ubiquitin modification that conveys this endocytosis-promoting signal is added to the receptor’s first cytoplasmic loop, catalyzed by the Smurf2 ubiquitin ligase, and coordinated with activation-dependent receptor phosphorylation and clustering through Smurf2 recruitment by the endocytic adaptor beta-arrestin. Epsin1 detects the signal at the CCP and is required for ubiquitin-promoted scission. This cargo-to-coat communication system mediates a biochemical checkpoint that ensures appropriate receptor ubiquitination for later trafficking, and it controls specific receptor loading into CCPs by sensing when a sufficient quorum is reached.Video

The First Five Seconds in the Life of a Clathrin-Coated Pit

Coated pits assemble by growth of a clathrin lattice, which is linked by adaptors to the underlying membrane. How does this process start? We used live-cell TIRF imaging with single-molecule EGFP sensitivity and high temporal resolution to detect arrival of the clathrin triskelions and AP2 adaptors that initiate coat assembly. Unbiased object identification and trajectory tracking, together with a statistical model, yield the arrival times and numbers of individual proteins, as well as experimentally confirmed estimates of the extent of substitution of endogenous by expressed, fluorescently tagged proteins. Pits initiate by coordinated arrival of clathrin and AP2, which is usually detected as two sequential steps, each of one triskelion with two adaptors. PI-4,5-P2 is essential for initiation. The accessory proteins FCHo1/2 are not; instead, they are required for sustained growth. This objective picture of coated pit initiation also shows that methods outlined here will be broadly useful for studies of dynamic assemblies in living cells.

Role of Disorder in Clathrin Lattice Assembly

Assembly of clathrin lattices is mediated by assembly/adaptor proteins which contain domains that bind lipids or membrane bound cargo proteins, and clathrin binding domains (CBDs) that recruit clathrin. Here we characterize the interaction between clathrin and a large fragment of the CBD of the clathrin assembly protein AP180. Mutational, NMR chemical shift, and analytical ultracentrifugation analyses allowed us to precisely define two clathrin binding sites within this fragment, each of which is found to bind weakly to the N-terminal domain of the clathrin heavy chain (TD). The locations of the two clathrin binding sites are consistent with predictions from sequence alignments of previously identified clathrin binding elements and, by extension, indicate that the complete AP180 CBD contains ∼12 degenerate repeats, each containing a single clathrin binding site. Sequence and circular dichroism analyses have indicated that the AP180 CBD is predominantly unstructured and our NMR analyses confirm that this is largely the case for the AP180 fragment characterized here. Unexpectedly, unlike the many proteins which undergo binding coupled folding upon interaction with their binding partners, the AP180 fragment is similarly unstructured in its bound and free states. Instead, we find that this fragment exhibits localized beta turn-like structures at the two clathrin binding sites both when free and bound to clathrin. These observations are incorporated into a model in which weak binding by multiple, pre-structured clathrin binding elements regularly dispersed throughout a largely unstructured CBD allows efficient recruitment of clathrin to endocytic sites and dynamic assembly of the clathrin lattice.

Clathrin Self-Assembly into Polyhedral Cages Studied by Computer Simulations

Clathrin is a three-legged proteins that self-assembles into polyhedral cages with regulatory and mechanical functions in the formation of cargo-laden vesicles at the cell-membrane and trans Golgi network. The essential features of self-assembly are innate to clathrin, as cages are also formed in purified slightly acid solutions. Our simulations of this process using a highly coarse-grained clathrin model reveal that a non-uniform distribution of interactions over clathrin's surface, rather than its characteristic shape, holds the key to self-assembly [1]. The cages are polydisperse, with a strong preference for a small subset of all possible configurations with twelve pentagonal and a variable number of hexagonal faces. Based on the experimental critical assembly concentration, we deduce an average binding energy of ∼23kT per clathrin [2]. The simulations also answer the long-standing question of how a flat purely hexagonal clathrin lattice can produce a cage with twelve pentagons: the introduction of spontaneous curvature through a change of hub and/or knee puckers causes tensions that result in the release of dome-shaped fragments, which may subsequently grow into full cages by recruiting cytosolic clathrin [2].[1] Biophys. J. 99, 1231 (2010).[2] Traffic 12, 1407 (2011).View Large Image | View Hi-Res Image | Download PowerPoint Slide

Clathrin light chain directs endocytosis by influencing the binding of the yeast Hip1R homologue, Sla2, to F-actin

The role of clathrin light chain (CLC) in clathrin-mediated endocytosis is not completely understood. Previous studies showed that the CLC N-terminus (CLC-NT) binds the Hip1/Hip1R/Sla2 family of membrane/actin-binding factors and that overexpression of the CLC-NT in yeast suppresses endocytic defects of clathrin heavy-chain mutants. To elucidate the mechanistic basis for this suppression, we performed synthetic genetic array analysis with a clathrin CLC-NT deletion mutation (clc1-Δ19-76). clc1-Δ19-76 suppressed the internalization defects of null mutations in three late endocytic factors: amphiphysins (rvs161 and rvs167) and verprolin (vrp1). In actin sedimentation assays, CLC binding to Sla2 inhibited Sla2 interaction with F-actin. Furthermore, clc1-Δ19-76 suppression of the rvs and vrp phenotypes required the Sla2 actin-binding talin-Hip1/R/Sla2 actin-tethering C-terminal homology domain, suggesting that clc1-Δ19-76 promotes internalization by prolonging actin engagement by Sla2. We propose that CLC directs endocytic progression by pruning the Sla2-actin attachments in the clathrin lattice, providing direction for membrane internalization.

Actin dynamics counteract membrane tension during clathrin-mediated endocytosis

Clathrin-mediated endocytosis is independent of actin dynamics in many circumstances but requires actin polymerization in others. We show that membrane tension determines the actin dependence of clathrin-coat assembly. As found previously, clathrin assembly supports formation of mature coated pits in the absence of actin polymerization on both dorsal and ventral surfaces of non-polarized mammalian cells, and also on basolateral surfaces of polarized cells. Actin engagement is necessary, however, to complete membrane deformation into a coated pit on apical surfaces of polarized cells and, more generally, on the surface of any cell in which the plasma membrane is under tension from osmotic swelling or mechanical stretching. We use these observations to alter actin dependence experimentally and show that resistance of the membrane to propagation of the clathrin lattice determines the distinction between "actin-dependent" and "actin-independent". We also find that light-chain bound Hip1R mediates actin engagement. These data thus provide a unifying explanation for the role of actin dynamics in coated-pit budding.

The Generation of Curved Clathrin Coats from Flat Plaques

Flat clathrin lattices or 'plaques' are commonly believed to be the precursors to clathrin-coated buds and vesicles. The sequence of steps carrying the flat hexagonal lattice into a highly curved polyhedral cage with exactly 12 pentagons remains elusive, however, and the large numbers of disrupted interclathrin connections in previously proposed conversion pathways make these scenarios rather unlikely. The recent notion that clathrin can make controlled small conformational transitions opens new avenues. Simulations with a self-assembling clathrin model suggest that localized conformational changes in a plaque can create sufficiently strong stresses for a dome-like fragment to break apart. The released fragment, which is strongly curved but still hexagonal, may subsequently grow into a cage by recruiting free triskelia from the cytoplasm, thus building all 12 pentagonal faces without recourse to complex topological changes. The critical assembly concentration in a slightly acidic in vitro solution is used to estimate the binding energy of a cage at 25-40 k(B) T/clathrin.

Membrane Deformation Caused by Clathrin and Associated Adaptor Proteins In Vitro

Clathrin-dependent endocytosis is a common mode of cellular trafficking. The formation of clathrin-coated vesicles is preceded by deformation of membrane due to the interacting coat proteins comprising of clathrin and various adaptors. However, the precise mechanism behind the deformation of membrane and existence of diverse shapes, sizes and symmetry remains unknown. We intend to develop a bio-mimetic system to allow the study of the precise biochemical and physical parameters that control the rate of clathrin polymerization and to determine the membrane deforming forces. Clathrin binding to giant unilamellar vesicles was followed using fluorescent clathrin and the coat polymerization was confirmed by fluorescent recovery after photobleaching (FRAP). Interestingly, clathrin binding and polymerization on membrane takes place only when AP180 is present in the injected mixture of proteins. We also observed significant differences in the membrane deformation caused by various adaptor proteins (AP180 and AP 1, 2) and clathrin by differential interference contrast fast video microscopy (DICM), on a micron scale. Additionally, interesting insights on the topology of the membrane deformations caused by various adaptor/clathrin mixtures on membrane sheet assemblies were obtained by means of atomic force microscopy (AFM). Our findings suggest that AP180/clathrin lead to the generation of extensive bud like structures that are ∼100nm in diameter and ∼20nm in height. Further, high resolution imaging will be used to differentiate various polygonal transformations in clathrin lattices under varying conditions of adaptor/clathrin, which may define various shapes and sizes of buds. In addition, precise forces and actors generating the forces behind membrane deformation and the role of pre-induced membrane curvature in the clathrin polymerization and varying lattice structure will be investigated.

Nanomechanics of Clathrin Protein Shells

The nanomechanical properties of protein shells, ranging from virus capsids to clathrin-coated vesicles, are functionally important and of notable recent interest. Using atomic force microscopy (AFM) and, more recently, quartz crystal microbalance-dissipation (QCM-D) instrumentation, we investigate mechanical properties of intact clathrin-coated vesicles (CCVs), reassembled pure clathrin cage baskets, and single triskelia. CCVs are purified subcellular trafficking organelles of about 100 nm diameter, consisting of an outer clathrin lattice shell over a receptor- and adaptor- loaded lipid vesicle. The clathrin cages formed by self-assembly of clathrin triskelia are structurally similar to the outer shell of CCVs. From AFM and complementary QCM-D measurements, we now provide an integrative mechanical model of the clathrin protein shell and of the coupling between the clathrin coat and the membrane vesicle, in biologically relevant fluid environments. Whereas the Young's modulus of the triskelia arm is as high as a few MPa, the effective shear moduli of clathrin triskelia, clathrin baskets, and CCVs are one to two orders of magnitude smaller and vary with the buffer pH and ionic strength, as well as their proximity to a given substrate support. Such modulation of the nanomechanical properties of the assembling/disassembling protein shells may be an essential aspect of cellular function.

Theoretical Modeling of the Weaving of Clathrin into Nanoscale Baskets

Many biological systems are capable of spontaneously assembling a diverse set of molecular architectures from a single subunit, without the need to pre-pattern the assembly. Cellular uptake of external substances is accomplished by a highly adaptive endocytosis process that accommodates a wide range of cargo shapes and sizes. Clathrin-mediated endocytosis involves the formation of a pit that is surrounded by a honeycomb coating whose pinwheel-shaped subunit is a clathrin-protein complex. We develop a theoretical model for the thermodynamics and kinetics of clathrin assembly, addressing the behavior in 2 and 3 dimensions, relevant to membrane and bulk assembly, respectively. The clathrin triskelions are modeled as effective flexible pinwheels that form leg-leg associations and resist elastic deformation. Thus, the pinwheels are capable of forming a range of ring structures, including 5-, 6-, and 7-member rings that are observed experimentally. Our theoretical model employs Monte Carlo simulations to address thermodynamic behavior and Brownian dynamics simulations to track the motion of clathrin pinwheels at sufficiently long time scales to achieve complete assembly. With this theoretical model, we predict the phase diagram for clathrin assembly incorporating binding interactions, elastic deformation, and defect-pair coupling, utilizing Kosterlitz-Thouless theory of defect-induced melting in 2 dimensions. Using analytical theory and computational simulations, we explore the role of binding strength and clathrin elasticity in the ability for clathrin lattices to dynamically reorganize due to local changes in membrane elasticity and tension. We then proceed to discuss the dynamics of lattice reorganization during the process of a clathrin-coated membrane wrapping around a nanoscale cargo.

Clathrin Self-Assembly into Polyhedral Cages by Computer Simulations

Clathrins are three-legged proteins that self-assemble into polyhedral cages with important regulatory and mechanical functions in the formation of cargo-laden vesicles at the cell-membrane (endocytosis) and the trans Golgi complex. The essential features of self-assembly are innate to clathrins, as cages are also formed in purified slightly acid solutions. This bare-bones process has been simulated here for the first time, revealing that an asymmetric distribution of interactions along the leg's circumference, rather than clathrin's characteristic shape, holds the key to the self-assembly process. The global puckering of the triskelion determines the average cage size, while the distribution of pentagonal and hexagonal facets in the self-assembled cages follows a simple selection rule. Simulations of planar clathrin lattices indicate that the introduction of spontaneous curvature, through a change of the clathrins's puckers, does not make a plaque curl up into a cage, but instead the plaque releases dome-shaped fragments which may subsequently grow into cages by recruiting cytosolic clathrins.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Single-molecule analysis of a molecular disassemblase reveals the mechanism of Hsc70-driven clathrin uncoating

Heat shock cognate protein 70 (Hsc70) supports remodeling of protein complexes -- for example, disassembly of clathrin coats on endocytic coated vesicles. To understand how a simple ATP driven molecular clamp catalyzes a large-scale disassembly reaction, we have used single-particle fluorescence imaging to track the dynamics of Hsc70 and its clathrin substrate in real time. Hsc70 accumulates to a critical level, determined by kinetic modeling to be one Hsc70 for every two functional attachment sites; rapid, all-or-none uncoating then ensues. We propose that Hsc70 traps conformational distortions, seen previously by electron cryomicroscopy, in the vicinity of each occupied site and that accumulation of local strains destabilises the clathrin lattice. Capture of conformational fluctuations may be a general mechanism for chaperone-driven disassembly of protein complexes.

<em>In vivo</em> and <em>in vitro</em> Studies of Adaptor-clathrin Interaction

A major endocytic pathway initiates with the formation of clathrin-coated vesicles (CCVs) that transport cargo from the cell surface to endosomes1-6. CCVs are distinguished by a polyhedral lattice of clathrin that coats the vesicle membrane and serves as a mechanical scaffold. Clathrin coats are assembled during vesicle formation from individual clathrin triskelia , the soluble form of clathrin composed of three heavy and three light chain subunits7,8. Because the triskelion does not have the ability to bind to the membrane directly, clathrin-binding adaptors are critical to link the forming clathrin lattice to the membrane through association with lipids and/or membrane proteins9. Adaptors also package transmembrane protein cargo, such as receptors, and can interact with each other and with other components of the CCV formation machinery9. Over twenty clathrin adaptors have been described, several are involved in clathrin mediated endocytosis and others localize to the trans Golgi network or endosomes9. With the exception of HIP1R (yeast Sla2p), all known clathrin adaptors bind to the N-terminal -propeller domain of the clathrin heavy chain9. Clathrin adaptors are modular proteins consisting of folded domains connected by unstructured flexible linkers. Within these linker regions, short binding motifs mediate interactions with the clathrin N-terminal domain or other components of the vesicle formation machinery9. Two distinct clathrin-binding motifs have been defined: the clathrin-box and the W-box9. The consensus clathrin-box sequence was originally defined as L[L/I][D/E/N][L/F][D/E]10 but variants have been subsequently discovered11. The W-box conforms to the sequence PWxxW (where x is any residue). Sla1p (Synthetic Lethal with Actin binding protein-1) was originally identified as an actin associated protein and is necessary for normal actin cytoskeleton structure and dynamics at endocytic sites in yeast cells12. Sla1p also binds the NPFxD endocytic sorting signal and is critical for endocytosis of cargo bearing the NPFxD signal13,14. More recently, Sla1p was demonstrated to bind clathrin through a motif similar to the clathrin box, LLDLQ, termed a variant clathrin-box (vCB), and to function as an endocytic clathrin adaptor15. In addition, Sla1p has become a widely used marker for the endocytic coat in live cell fluorescence microscopy studies16. Here we use Sla1p as a model to describe approaches for adaptor-clathrin interaction studies. We focus on live cell fluorescence microscopy, GST-pull down, and co-immunoprecipitation methods.

<em>In vivo</em> and <em>in vitro</em> Studies of Adaptor-clathrin Interaction

A major endocytic pathway initiates with the formation of clathrin-coated vesicles (CCVs) that transport cargo from the cell surface to endosomes. CCVs are distinguished by a polyhedral lattice of clathrin that coats the vesicle membrane and serves as a mechanical scaffold. Clathrin coats are assembled during vesicle formation from individual clathrin triskelia , the soluble form of clathrin composed of three heavy and three light chain subunits. Because the triskelion does not have the ability to bind to the membrane directly, clathrin-binding adaptors are critical to link the forming clathrin lattice to the membrane through association with lipids and/or membrane proteins. Adaptors also package transmembrane protein cargo, such as receptors, and can interact with each other and with other components of the CCV formation machinery. Over twenty clathrin adaptors have been described, several are involved in clathrin mediated endocytosis and others localize to the trans Golgi network or endosomes. With the exception of HIP1R (yeast Sla2p), all known clathrin adaptors bind to the N-terminal -propeller domain of the clathrin heavy chain. Clathrin adaptors are modular proteins consisting of folded domains connected by unstructured flexible linkers. Within these linker regions, short binding motifs mediate interactions with the clathrin N-terminal domain or other components of the vesicle formation machinery. Two distinct clathrin-binding motifs have been defined: the clathrin-box and the W-box. The consensus clathrin-box sequence was originally defined as L[L/I][D/E/N][L/F][D/E] but variants have been subsequently discovered. The W-box conforms to the sequence PWxxW (where x is any residue). Sla1p (Synthetic Lethal with Actin binding protein-1) was originally identified as an actin associated protein and is necessary for normal actin cytoskeleton structure and dynamics at endocytic sites in yeast cells. Sla1p also binds the NPFxD endocytic sorting signal and is critical for endocytosis of cargo bearing the NPFxD signal. More recently, Sla1p was demonstrated to bind clathrin through a motif similar to the clathrin box, LLDLQ, termed a variant clathrin-box (vCB), and to function as an endocytic clathrin adaptor. In addition, Sla1p has become a widely used marker for the endocytic coat in live cell fluorescence microscopy studies. Here we use Sla1p as a model to describe approaches for adaptor-clathrin interaction studies. We focus on live cell fluorescence microscopy, GST-pull down, and co-immunoprecipitation methods.

Impact of defect creation and motion on the thermodynamics and large-scale reorganization of self-assembled clathrin lattices

We develop a theoretical model for the thermodynamics and kinetics of clathrin self-assembly. Our model addresses the behavior in two dimensions and can be easily extended to three dimensions, facilitating the study of membrane, surface, and bulk assembly. The clathrin triskelia are modeled as flexible pinwheels that form leg-leg associations and resist bending and stretching deformations. Thus, the pinwheels are capable of forming a range of ring structures, including 5-, 6-, and 7-member rings that are observed experimentally. Our theoretical model employs Brownian dynamics to track the motion of clathrin pinwheels at sufficiently long time scales to achieve complete assembly. Invoking theories of dislocation-mediated melting in two dimensions, we discuss the phase behavior for clathrin self-assembly as predicted by our theoretical model. We demonstrate that the generation of 5–7 defects in an otherwise perfect honeycomb lattice resembles creation of two dislocations with equal and opposite Burgers vectors. We use orientational- and translational-order correlation functions to predict the crystalline-hexatic and hexatic-liquid phase transitions in clathrin lattices. These results illustrate the pivotal role that molecular elasticity plays in the physical behavior of self-assembling and self-healing materials.

Dynamic Interactions between Clathrin and Locally Structured Elements in a Disordered Protein Mediate Clathrin Lattice Assembly

Assembly of clathrin lattices is mediated by assembly/adaptor proteins that contain domains that bind lipids or membrane-bound cargo proteins and clathrin binding domains (CBDs) that recruit clathrin. Here, we characterize the interaction between clathrin and a large fragment of the CBD of the clathrin assembly protein AP180. Mutational, NMR chemical shift, and analytical ultracentrifugation analyses allowed us to precisely define two clathrin binding sites within this fragment, each of which is found to bind weakly to the N-terminal domain of the clathrin heavy chain (TD). The locations of the two clathrin binding sites are consistent with predictions from sequence alignments of previously identified clathrin binding elements and, by extension, indicate that the complete AP180 CBD contains ∼ 12 degenerate repeats, each containing a single clathrin binding site. Sequence and circular dichroism analyses have indicated that the AP180 CBD is predominantly unstructured and our NMR analyses confirm that this is largely the case for the AP180 fragment characterized here. Unexpectedly, unlike the many proteins that undergo binding-coupled folding upon interaction with their binding partners, the AP180 fragment is similarly unstructured in its bound and free states. Instead, we find that this fragment exhibits localized β-turn-like structures at the two clathrin binding sites both when free and when bound to clathrin. These observations are incorporated into a model in which weak binding by multiple, pre-structured clathrin binding elements regularly dispersed throughout a largely unstructured CBD allows efficient recruitment of clathrin to endocytic sites and dynamic assembly of the clathrin lattice.