Tandem Calponin Homology Research Articles

\u03b2-III-spectrin N-terminus is required for high-affinity actin binding and SCA5 neurotoxicity

Recent structural studies of β-III-spectrin and related cytoskeletal proteins revealed N-terminal sequences that directly bind actin. These sequences are variable in structure, and immediately precede a conserved actin-binding domain composed of tandem calponin homology domains (CH1 and CH2). Here we investigated in Drosophila the significance of the β-spectrin N-terminus, and explored its functional interaction with a CH2-localized L253P mutation that underlies the neurodegenerative disease spinocerebellar ataxia type 5 (SCA5). We report that pan-neuronal expression of an N-terminally truncated β-spectrin fails to rescue lethality resulting from a β-spectrin loss-of-function allele, indicating that the N-terminus is essential to β-spectrin function in vivo. Significantly, N-terminal truncation rescues neurotoxicity and defects in dendritic arborization caused by L253P. In vitro studies show that N-terminal truncation eliminates L253P-induced high-affinity actin binding, providing a mechanistic basis for rescue. These data suggest that N-terminal sequences may be useful therapeutic targets for small molecule modulation of the aberrant actin binding associated with SCA5 β-spectrin and spectrin-related disease proteins.

Structure and Function of Abnormal Spindle-Like, Microtubule Associated Protein (ASPM)

Human autosomal recessive primary microcephaly (MCPH) is a hereditary disorder associated with a small brain and associated intellectual disability. Mutations in the gene for Abnormal Spindle-like, Microtubule Associated protein (ASPM) are the most common causes of MCPH. ASPM, a large (∼400kDa) protein, is important in mitosis. It anchors the mitotic spindle at the spindle poles and is essential for normal cytokinesis. However, the complete structure-function of ASPM is not well understood.The N-terminal domain of ASPM contains 2 tandem calponin homology (CH) domains and binds microtubules. However the location of the specific binding site is unclear. Two tandem CH domains typically bind actin, whereas single CH domains bind microtubules suggesting the CH domains of ASPM bind actin. The region following the CH domains contains 81 isoleucine-glutamine (IQ) motifs, known to bind light chains such as calmodulin. These motifs form a single alpha-helix to which the light chains bine, suggesting an overall elongated structure for ASPM.Using a combination of mass spectrometry and flow cell assays, we determined that the tandem CH domains of ASPM bind to microtubules, and not actin, suggesting that the CH domains may be the key binding site of ASPM for microtubules within the N-terminus. Negative stain electron microscopy of a central 21 IQ motif-containing domain revealed a long semi-flexible molecule, fully decorated with light chains. These data thus start to reveal the structure and function of ASPM.

Biased localization of actin binding proteins by actin filament conformation

The assembly of actin filaments into distinct cytoskeletal structures plays a critical role in cell physiology, but how proteins localize differentially to these structures within a shared cytoplasm remains unclear. Here, we show that the actin-binding domains of accessory proteins can be sensitive to filament conformational changes. Using a combination of live cell imaging and in vitro single molecule binding measurements, we show that tandem calponin homology domains (CH1–CH2) can be mutated to preferentially bind actin networks at the front or rear of motile cells. We demonstrate that the binding kinetics of CH1–CH2 domain mutants varies as actin filament conformation is altered by perturbations that include stabilizing drugs and other binding proteins. These findings suggest that conformational changes of actin filaments in cells could help to direct accessory binding proteins to different actin cytoskeletal structures through a biophysical feedback loop.

Tissue-Specificity of Dystrophin-Actin Interactions: Isoform-Specific Thermodynamic Stability and Actin-Binding Function of Tandem Calponin-Homology Domains.

Genetic mutations in Duchenne muscular dystrophy (DMD) gene affecting the expression of dystrophin protein lead to a number of muscle disorders collectively called dystrophinopathies. In addition to muscle dystrophin, mutations in brain-specific dystrophin isoforms, in particular those that are expressed in the brain cortex and Purkinje neurons, result in cognitive impairment associated with DMD. These isoforms carry minor variations in the flanking region of the N-terminal actin-binding domain (ABD1) of dystrophin, which is composed of two calponin-homology (CH) domains in tandem. Determining the effect of these sequence variations is critical for understanding the mechanisms that govern varied symptoms of the disease. We studied the impact of differences in the N-terminal flanking region on the structure and function of dystrophin tandem CH domain isoforms. The amino acid changes did not affect the global structure of the protein but drastically affected the thermodynamic stability, with the muscle isoform more stable than the brain and Purkinje isoforms. Actin binding investigated with actin from different sources (skeletal muscle, smooth muscle, cardiac muscle, and platelets) revealed that the muscle isoform binds to filamentous actin (F-actin) with a lower affinity compared to the brain and Purkinje isoforms, and a similar trend was observed with actin from different sources. In addition, all isoforms showed a higher affinity to smooth muscle actin in comparison to actin from other sources. In conclusion, tandem CH domain isoforms might be using minor sequence variations in the N-terminal flanking regions to modulate their thermodynamic stability and actin-binding function, thus leading to specificity in dystrophin–actin interactions in various tissues.

Steric regulation of tandem calponin homology domain actin-binding affinity.

Tandem calponin homology (CH1–CH2) domains are common actin-binding domains in proteins that interact with and organize the actin cytoskeleton. Despite regions of high sequence similarity, CH1–CH2 domains can have remarkably different actin-binding properties, with disease-associated point mutants known to increase as well as decrease affinity for F-actin. To investigate features that affect CH1–CH2 affinity for F-actin in cells and in vitro, we perturbed the utrophin actin-binding domain by making point mutations at the CH1–CH2 interface, replacing the linker domain, and adding a polyethylene glycol (PEG) polymer to CH2. Consistent with a previous model describing CH2 as a steric negative regulator of actin binding, we find that utrophin CH1–CH2 affinity is both increased and decreased by modifications that change the effective “openness” of CH1 and CH2 in solution. We also identified interface mutations that caused a large increase in affinity without changing solution “openness,” suggesting additional influences on affinity. Interestingly, we also observe nonuniform subcellular localization of utrophin CH1–CH2 that depends on the N-terminal flanking region but not on bulk affinity. These observations provide new insights into how small sequence changes, such as those found in diseases, can affect CH1–CH2 binding properties.

Structural basis of the filamin A actin-binding domain interaction with F-actin.

Actin-crosslinking proteins assemble actin filaments into higher-order structures essential for orchestrating cell shape, adhesion and motility. Missense mutations in the tandem calponin homology (CH) domains of their actin-binding domains (ABDs) underlie numerous genetic diseases, but a molecular understanding of these pathologies is hampered by the lack of high-resolution structures of any actin-crosslinking protein bound to F-actin. Here, taking advantage of a high-affinity, disease-associated mutant of the human filamin A (FLNa) ABD, we combine cryo-electron microscopy and functional studies to reveal at near-atomic resolution how the first CH domain (CH1) and residues immediately N-terminal to it engage actin. We further show that reorientation of CH2 relative to CH1 is required to avoid clashes with actin and to expose F-actin-binding residues on CH1. Our data explain localization of disease-associated loss-of-function mutations to FLNaCH1 and gain-of-function mutations to the regulatory FLNaCH2. Sequence conservation argues that this provides a general model for ABD-F-actin binding.

The N-Terminal Flanking Region Modulates the Actin Binding Affinity of the Utrophin Tandem Calponin-Homology Domain.

Despite sharing a high degree of sequence similarity, the tandem calponin-homology (CH) domain of utrophin binds to actin 30 times stronger than that of dystrophin. We have previously shown that this difference in actin binding affinity could not be ascribed to the differences in inter-CH-domain linkers [Bandi, S., et al. (2015) Biochemistry 54, 5480-5488]. Here, we examined the role of the N-terminal flanking region. The utrophin tandem CH domain contains a 27-residue flanking region before its CH1 domain. We examined its effect by comparing the structure and function of full-length utrophin tandem CH domain Utr(1-261) and its truncated Utr(28-261) construct. Both full-length and truncated constructs are monomers in solution, with no significant differences in their secondary or tertiary structures. Truncated construct Utr(28-261) binds to actin 30 times weaker than that of the full-length Utr(1-261), similar to that of the dystrophin tandem CH domain with a much shorter flanking region. Deletion of the N-terminal flanking region stabilizes the CH1 domain. The magnitude of the change in binding free energy upon truncation is similar to that of the change in thermodynamic stability. The isolated N-terminal peptide by itself is significantly random coil and does not bind to F-actin in the affinity range of Utr(1-261) and Utr(28-261). These results indicate that the N-terminal flanking region significantly affects the actin binding affinity of tandem CH domains. This observation further stresses that protein regions other than the three actin-binding surfaces identified earlier, irrespective of whether they directly bind to actin, also contribute to the actin binding affinity of tandem CH domains.

A human β-III-spectrin spinocerebellar ataxia type 5 mutation causes high-affinity F-actin binding.

Spinocerebellar ataxia type 5 (SCA5) is a human neurodegenerative disease that stems from mutations in the SPTBN2 gene encoding the protein β-III-spectrin. Here we investigated the molecular consequence of a SCA5 missense mutation that results in a L253P substitution in the actin-binding domain (ABD) of β-III-spectrin. We report that the L253P substitution in the isolated β-III-spectrin ABD causes strikingly high F-actin binding affinity (Kd = 75.5 nM) compared to the weak F-actin binding affinity of the wild-type ABD (Kd = 75.8 μM). The mutation also causes decreased thermal stability (Tm = 44.6 °C vs 59.5 °C). Structural analyses indicate that leucine 253 is in a loop at the interface of the tandem calponin homology (CH) domains comprising the ABD. Leucine 253 is predicted to form hydrophobic contacts that bridge the CH domains. The decreased stability of the mutant indicates that these bridging interactions are probably disrupted, suggesting that the high F-actin binding affinity of the mutant is due to opening of the CH domain interface. These results support a fundamental role for leucine 253 in regulating opening of the CH domain interface and binding of the ABD to F-actin. This study indicates that high-affinity actin binding of L253P β-III-spectrin is a likely driver of neurodegeneration.

The N- and C-Terminal Domains Differentially Contribute to the Structure and Function of Dystrophin and Utrophin Tandem Calponin-Homology Domains.

Dystrophin and utrophin are two muscle proteins involved in Duchenne/Becker muscular dystrophy. Both proteins use tandem calponin-homology (CH) domains to bind to F-actin. We probed the role of N-terminal CH1 and C-terminal CH2 domains in the structure and function of dystrophin tandem CH domain and compared with our earlier results on utrophin to understand the unifying principles of how tandem CH domains work. Actin cosedimentation assays indicate that the isolated CH2 domain of dystrophin weakly binds to F-actin compared to the full-length tandem CH domain. In contrast, the isolated CH1 domain binds to F-actin with an affinity similar to that of the full-length tandem CH domain. Thus, the obvious question is why the dystrophin tandem CH domain requires CH2, when its actin binding is determined primarily by CH1. To answer, we probed the structural stabilities of CH domains. The isolated CH1 domain is very unstable and is prone to serious aggregation. The isolated CH2 domain is very stable, similar to the full-length tandem CH domain. These results indicate that the main role of CH2 is to stabilize the tandem CH domain structure. These conclusions from dystrophin agree with our earlier results on utrophin, indicating that this phenomenon of differential contribution of CH domains to the structure and function of tandem CH domains may be quite general. The N-terminal CH1 domains primarily determine the actin binding function whereas the C-terminal CH2 domains primarily determine the structural stability of tandem CH domains, and the extent of stabilization depends on the strength of inter-CH domain interactions.

Interdomain Linker Determines Primarily the Structural Stability of Dystrophin and Utrophin Tandem Calponin-Homology Domains Rather than Their Actin-Binding Affinity.

Tandem calponin-homology (CH) domains are the most common actin-binding domains in proteins. However, structural principles underlying their function are poorly understood. These tandem domains exist in multiple conformations with varying degrees of inter-CH-domain interactions. Dystrophin and utrophin tandem CH domains share high sequence similarity (∼82%), yet differ in their structural stability and actin-binding affinity. We examined whether the conformational differences between the two tandem CH domains can explain differences in their stability and actin binding. Dystrophin tandem CH domain is more stable by ∼4 kcal/mol than that of utrophin. Individual CH domains of dystrophin and utrophin have identical structures but differ in their relative orientation around the interdomain linker. We swapped the linkers between dystrophin and utrophin tandem CH domains. Dystrophin tandem CH domain with utrophin linker (DUL) has similar stability as that of utrophin tandem CH domain. Utrophin tandem CH domain with dystrophin linker (UDL) has similar stability as that of dystrophin tandem CH domain. Dystrophin tandem CH domain binds to F-actin ∼30 times weaker than that of utrophin. After linker swapping, DUL has twice the binding affinity as that of dystrophin tandem CH domain. Similarly, UDL has half the binding affinity as that of utrophin tandem CH domain. However, changes in binding free energies due to linker swapping are much lower by an order of magnitude compared to the corresponding changes in unfolding free energies. These results indicate that the linker region determines primarily the structural stability of tandem CH domains rather than their actin-binding affinity.

Rare Example of a Protein where an Isolated Domain is More Stable than the Full-Length

Studying the contribution of individual domains to protein structure and function is of considerable interest over the years. Domains are in general less stable than the corresponding full-length proteins. Here, we report an exceptional case of utrophin tandem calponin-homology (CH) domain. Isolated C-terminal CH domain (CH2) is both thermodynamically and kinetically more stable than the full-length tandem CH domain. Reversible, equilibrium denaturant melts using both circular dichroism and protein fluorescence signals show that the CH2 is thermodynamically more stable by 4.0 kcal/mol when compared with the full-length tandem CH domain. Thermal melts indicate that CH2 unfolds at a higher temperature (15°C) than the full-length protein. Stopped-flow kinetics indicate that the CH2 unfolds slower (by 3 times) and folds faster (by 7 times) than the full-length protein, suggesting the higher kinetic stability of CH2. Analytical ultracentrifugation, size-exclusion chromatography, and dynamic light scattering show that both CH2 and the full-length protein are monomers in solution, confirming that the higher stability of CH2 is not due to formation of oligomers. Thus, the utrophin tandem CH domain is a rare example in which an isolated domain is more stable than the corresponding full-length protein.

Abstract 42: Differential Contribution Of The N- And C-terminal Domains To The Folding And Function Of Tandem Calponin-homology Domains

Tandem calponin-homology (CH) domains constitute a major class of actin-binding domains that include dystrophin and utrophin, the two key proteins involved in muscular dystrophy. Despite their importance, how their structure controls their function is not understood. Here, we study the contribution of individual CH domains to the actin-binding function and thermodynamic stability of utrophin’s tandem CH domain. Traditional actin co-sedimentation assays indicate that the isolated C-terminal CH2 domain binds weakly to F-actin when compared with the full-length tandem CH domain. In contrast, isolated CH1 binds to F-actin with a similar efficiency as that of the full-length tandem CH domain. Thus, the obvious question that arises is why tandem CH domains require CH2, when their actin-binding efficiency is originating primarily from CH1. To answer, we probed the thermodynamic stabilities of individual CH domains. Isolated CH1 domain is unstable and is prone to serious aggregation. Isolated CH2 is very stable, even appears to be more stable than the full-length tandem CH domain. In addition, the CH2 domain, which is more stable, is less functional. These results indicate that the main function of CH2 is to stabilize CH1. Consistently, the proposed structure of utrophin’s tandem CH domain based on earlier X-ray studies indicates a close proximity between the C-terminal helix of CH2 and the N-terminal helix of CH1, and this helix in CH2 is more dynamic in the full-length protein when compared with that in the absence of CH1, suggesting the mechanism by which CH2 stabilizes CH1. These observations indicate that the two CH domains contribute differentially to the folding and function of tandem CH domains, although both domains essentially have the same native structure in the tandem CH domain. The N-terminal domain determines the function, whereas the C-terminal domain determines the stability. This work was funded by the AHA Grant 11SDG4880046.

The C-Terminal Domain of the Utrophin Tandem Calponin-Homology Domain Appears To Be Thermodynamically and Kinetically More Stable Than the Full-Length Protein

Domains are in general less stable than the corresponding full-length proteins. Human utrophin tandem calponin-homology (CH) domain seems to be an exception. Reversible, equilibrium denaturant melts indicate that the isolated C-terminal domain (CH2) is thermodynamically more stable than the tandem CH domain. Thermal melts show that CH2 unfolds at a temperature higher than that at which the full-length protein unfolds. Stopped-flow kinetics indicates that CH2 unfolds slower than the full-length protein, indicating its higher kinetic stability. Thus, the utrophin tandem CH domain may be one of the few proteins in which an isolated domain is more stable than the corresponding full-length protein.

The Actin Binding Affinity of the Utrophin Tandem Calponin-Homology Domain Is Primarily Determined by Its N-Terminal Domain

The structural determinants of the actin binding function of tandem calponin-homology (CH) domains are poorly understood, particularly the role of individual domains. We determined the actin binding affinity of isolated CH domains from human utrophin and compared them with the affinity of the full-length tandem CH domain. Traditional cosedimentation assays indicate that the C-terminal CH2 domain binds to F-actin much weaker than the full-length tandem CH domain. The N-terminal CH1 domain is less stable and undergoes severe protein aggregation; therefore, traditional actin cosedimentation assays could not be used. To address this, we have developed a folding-upon-binding method. We refolded the CH1 domain from its unfolded state in the presence of F-actin. This results in a competition between actin binding and aggregation. A differential centrifugation technique was used to distinguish actin binding from aggregation. Low-speed centrifugation pelleted CH1 aggregates, but not F-actin or its bound protein. Subsequent high-speed centrifugation resulted in the cosedimentation of bound CH1 along with F-actin. The CH1 domain binds to F-actin with an affinity similar to that of the full-length tandem CH domain, unlike the CH2 domain. The actin binding cooperativity between the two domains was quantitatively calculated from the association constants of the full-length tandem CH domain and its CH domains, and found to be much smaller than the association constant of the CH1 domain alone. These results indicate that the actin binding affinity of the utrophin tandem CH domain is primarily determined by its CH1 domain, when compared to that of its CH2 domain or the cooperativity between the two CH domains.

Tug-Of-War Between Thermodynamic Stability and Actin-Binding Function of Tandem Calponin-Homology Domains

Tandem calponin-homology (CH) domains constitute a major class of actin-binding domains that include dystrophin and utrophin, the two key proteins involved in muscular dystrophy. Despite their importance, how their structure controls their function is not understood. Here, we study the contribution of individual CH domains to the actin-binding function of utrophin's tandem CH domain. Co-sedimentation assays indicate that the C-terminal CH2 domain binds weakly to F-actin when compared with the full-length tandem CH domain, consistent with the published results on tandem CH domains. However, the surprise came from the CH1 domain. Isolated CH1 binds strongly to F-actin when compared with the full-length tandem CH domain. These results indicate that CH2 has a negative influence on actin-binding when it is linked with CH1. Thus, the obvious question that arises is why tandem CH domains require CH2, when CH2 is reducing their actin-binding efficiency. To answer, we probed the thermodynamic stabilities of individual CH domains. Isolated CH1 domain is unstable and is prone to serious aggregation. Isolated CH2 is very stable, even more stable than that of the full-length tandem CH domain. This makes utrophin's tandem CH domain as the first example where an isolated domain is more stable than the full-length protein. These results indicate that the main function of CH2 is to stabilize CH1 at the expense of decreasing the actin-binding efficiency. Consistently, the proposed structure of utrophin's tandem CH domain based on earlier X-ray studies indicates a close proximity between the C-terminal helix of CH2 and the N-terminal helix of CH1, and this helix in CH2 becomes more dynamic in the full-length protein when compared with that in the absence of CH1, suggesting a mechanism by which CH2 stabilizes CH1 despite the decrease in actin-binding function.

Abstract 286: Structural Determinants Of Thermodynamic Stability And Actin-binding Function Of Dystrophin And Utrophin

Muscular dystrophy (MD) is an incurable disease, and affects all types of muscles. The decreased function of heart muscles causes heart problems such as cardiomyopathy and congestive heart failure. A deficiency in functional dystrophin protein in muscle cells triggers the onset of Duchenne MD and Becker MD. Utrophin is the closest homologue of dystrophin and is being tested as a protein drug to replace the loss of functional dystrophin in human patients. However, utrophin is less stable and binds to actin through a different mode of contact when compared with dystrophin. To optimize utrophin as the protein drug, we first need to understand how its molecular structure controls its stability and function. Utrophin and dystrophin bind to actin using tandem calponin-homology (CH) domains at their N-terminus. Individual CH domains of utrophin and dystrophin are very similar in amino acid sequence and their three dimensional structures. The major difference is in their relative orientation around the linker region that connects the two CH domains. To determine the role of the linker, we designed constructs with linkers switched between utrophin and dystrophin. Our results indicate that the tandem CH domain of utrophin with dystrophin linker (UDL) is more stable than that of utrophin but less stable than that of dystrophin. Similarly, tandem CH domain of dystrophin with utrophin linker (DUL) is less stable than that of dystrophin but more stable than that of utrophin. Kinetic folding and unfolding studies suggest that the linker predominantly affects the folding rate rather than the unfolding rate. In addition to stability and folding, the linker also controls protein aggregation. UDL is more prone to aggregation when compared with that of utrophin, whereas DUL is less prone to aggregation when compared with that of dystrophin. These results indicate that the linker plays a major role in controlling the stability and function of tandem CH domains, and further suggests that it is possible to engineer utrophin to behave precisely like dystrophin based on their molecular structures.

Structural Stability of Tandem Calponin-Homology (CH) Domains Originates from their C-Terminal CH2 Domain

Tandem CH domains form a major class of actin binding domains. In general, the N-terminal CH domain (CH1) weakly binds to F-actin whereas the C-terminal CH domain (CH2) does not bind to actin. However, when CH2 is linked to CH1, the actin binding efficiency increases by more than ten times, which implies a functional cooperativity between the two CH domains. The structural cooperativity underlying this functional cooperativity and the physical mechanism by which CH2 domain enhances the actin-binding efficiency is not understood. In this study, we examined the relative stabilities of the two CH domains of utrophin and dystrophin. The isolated CH1 domain of utrophin does not exist as a stable structure; it is more like a destabilized “molten globule” state. However, its CH2 domain folds to a stable structure, as evident from its alpha-helical spectrum and cooperative melt. Similar to utrophin, the CH2 domain of dystrophin is a well-structured protein, and has similar stability as that of the full-length tandem CH domain. The CH1 domain of dystrophin is quite unstable and aggregates severely. These results indicate that the CH1 domain requires CH2 for its folding, or in other words, CH2 acts as a template for CH1 folding. These stability experiments support the earlier hypothesis proposed based on cryo-EM studies that the major role of CH2 might be to stabilize the tandem CH domain. Additional support comes from the literature: no molecular structures are available for the CH1 domains alone, suggesting that they might be unstable in the absence of CH2. In contrast, structures have been determined for numerous CH2 domains. These experimental observations indicate that the CH2 domain enhances the actin-binding function by imparting structural stability to the tandem CH domains.

Dystrophin’s Tandem Calponin-Homology Domains: Is the Case Closed?

Dystrophin’s Tandem Calponin-Homology Domains: Is the Case Closed?

The N-Terminal Actin-Binding Tandem Calponin-Homology (CH) Domain of Dystrophin Is in a Closed Conformation in Solution and When Bound to F-actin

Deficiency of the vital muscle protein dystrophin triggers Duchenne/Becker muscular dystrophy, but the structure-function relationship of dystrophin is poorly understood. To date, molecular structures of three dystrophin domains have been determined, of which the N-terminal actin-binding domain (N-ABD or ABD1) is of particular interest. This domain is composed of two calponin-homology (CH) domains, which form an important class of ABDs in muscle proteins. A previously determined x-ray structure indicates that the dystrophin N-ABD is a domain-swapped dimer, with each monomer adopting an extended, open conformation in which the two CH domains do not interact. This structure is controversial because it contradicts functional studies and known structures of similar ABDs from other muscle proteins. Here, we investigated the solution conformation of the dystrophin N-ABD using a very simple and elegant technique of pyrene excimer fluorescence. Using the wild-type protein, which contains two cysteines, and the corresponding single-cysteine mutants, we show that the protein is a monomer in solution and is in a closed conformation in which the two CH domains seem to interact, as observed from the excimer fluorescence of pyrene-labeled wild-type protein. Excimer fluorescence was also observed in its actin-bound form, indicating that the dystrophin N-ABD binds to F-actin in a closed conformation. Comparison of the dystrophin N-ABD conformation with other ABDs indicates that the tandem CH domains in general may be monomeric in solution and predominantly occur in closed conformation, whereas their actin-bound conformations may differ.

Multimodal microtubule binding by the Ndc80 kinetochore complex

SummaryThe Ndc80 complex is a key site of kinetochore-microtubule attachment during cell division. The human complex engages microtubules with a globular “head” formed by tandem calponin-homology domains and an 80 amino-acid unstructured “tail” that contains sites of phospho-regulation by the Aurora B kinase. Using biochemical, cell biological, and electron microscopy analyses, we have dissected the tail’s roles in microtubule binding and mediating cooperative interactions between Ndc80 complexes. Two segments of the tail that contain Aurora B sites become ordered at interfaces; one with tubulin and the second with an adjacent Ndc80 head on the microtubule surface, forming interactions which are disrupted by phosphorylation. We propose a model in which Ndc80’s interaction with either growing or shrinking microtubule ends can be tuned by the phosphorylation state of its tail.