Molecular basis of Q-length selectivity for the MW1 antibody-huntingtin interaction.

Huntington's disease is caused by a polyglutamine (polyQ) expansion in the huntingtin protein. Huntingtin exon 1 (Httex1), as well as other naturally occurring N-terminal huntingtin fragments with expanded polyQ are prone to aggregation, forming potentially cytotoxic oligomers and fibrils. Antibodies and other N-terminal huntingtin binders are widely explored as biomarkers and possible aggregation-inhibiting therapeutics. A monoclonal antibody, MW1, is known to preferentially bind to huntingtin fragments with expanded polyQ lengths, but the molecular basis of the polyQ length specificity remains poorly understood. Using solution NMR, electron paramagnetic resonance, and other biophysical methods, we investigated the structural features of the Httex1-MW1 interaction. Rather than recognizing residual α-helical structure, which is promoted by expanded Q-lengths, MW1 caused the formation of a new, non-native, conformation in which the entire polyQ is largely extended. This non-native polyQ structure allowed the formation of large mixed Httex1-MW1 multimers (600-2900 kD), when Httex1 with pathogenic Q-length (Q46) was used. We propose that these multivalent, entropically favored interactions, are available only to proteins with longer Q-lengths and represent a major factor governing the Q-length preference of MW1. The present study reveals that it is possible to target proteins with longer Q-lengths without having to stabilize a natively favored conformation. Such mechanisms could be exploited in the design of other Q-length specific binders.

Comparison of Integrin αIIbß3 Transmembrane Association in Vesicles and Bicelles.

Membrane proteins are commonly reconstituted in membrane mimics exhibiting discontinuous lipid bilayers. In contrast, the continuous membranes of cells are conceptually best represented by large unilamellar vesicles (LUVs). Here, we compared the thermodynamic stability of the integrin αIIbß3 transmembrane (TM) complex between vesicles and bicelles to assess the consequence of this simplification. In LUVs, we further evaluated the strength of the αIIb(G972S)-ß3(V700T) interaction that corresponds to the hydrogen bond interaction postulated for ß2 integrins. An upper limit of 0.9 kcal/mol was estimated for superior TM complex stabilization in LUVs relative to bicelles. Compared to the αIIbß3 TM complex stability in LUVs of 5.6 ± 0.2 kcal/mol, this limit is modest, indicating that bicelles performed well relative to LUVs. The implementation of ß3(V700T) alleviated αIIb(G972S) destabilization by 0.4 ± 0.2 kcal/mol in confirmation of relatively weak hydrogen bonding. Interestingly, the hydrogen bond adjusts the TM complex stability to a level that is not achievable by merely varying the residue corresponding to αIIb(Gly972).

The folding equilibrium of huntingtin exon 1 monomer depends on its polyglutamine tract.

Expansion of the polyglutamine (polyQ) tract in exon 1 of the huntingtin protein (Httex1) leads to Huntington's disease resulting in fatal neurodegeneration. However, it remains poorly understood how polyQ expansions alter protein structure and cause toxicity. Using CD, EPR, and NMR spectroscopy, we found here that monomeric Httex1 consists of two co-existing structural states whose ratio is determined by polyQ tract length. We observed that short Q-lengths favor a largely random-coil state, whereas long Q-lengths increase the proportion of a predominantly α-helical state. We also note that by following a mobility gradient, Httex1 α-helical conformation is restricted to the N-terminal N17 region and to the N-terminal portion of the adjoining polyQ tract. Structuring in both regions was interdependent and likely stabilized by tertiary contacts. Although little helicity was present in N17 alone, each Gln residue in Httex1 enhanced helix stability by 0.03-0.05 kcal/mol, causing a pronounced preference for the α-helical state at pathological Q-lengths. The Q-length-dependent structuring and rigidification could be mimicked in proteins with shorter Q-lengths by a decrease in temperature, indicating that lower temperatures similarly stabilize N17 and polyQ intramolecular contacts. The more rigid α-helical state of Httex1 with an expanded polyQ tract is expected to alter interactions with cellular proteins and modulate the toxic Httex1 misfolding process. We propose that the polyQ-dependent shift in the structural equilibrium may enable future therapeutic strategies that specifically target Httex1 with toxic Q-lengths.

A Conserved Ectodomain-Transmembrane Domain Linker Motif Tunes the Allosteric Regulation of Cell Surface Receptors.

In many families of cell surface receptors, a single transmembrane (TM) α-helix separates ecto- and cytosolic domains. A defined coupling of ecto- and TM domains must be essential to allosteric receptor regulation but remains little understood. Here, we characterize the linker structure, dynamics, and resulting ecto-TM domain coupling of integrin αIIb in model constructs and relate it to other integrin α subunits by mutagenesis. Cellular integrin activation assays subsequently validate the findings in intact receptors. Our results indicate a flexible yet carefully tuned ecto-TM coupling that modulates the signaling threshold of integrin receptors. Interestingly, a proline at the N-terminal TM helix border, termed NBP, is critical to linker flexibility in integrins. NBP is further predicted in 21% of human single-pass TM proteins and validated in cytokine receptors by the TM domain structure of the cytokine receptor common subunit ß and its P441A-substituted variant. Thus, NBP is a conserved uncoupling motif of the ecto-TM domain transition and the degree of ecto-TM domain coupling represents an important parameter in the allosteric regulation of diverse cell surface receptors.

Annular anionic lipids stabilize the integrin αIIbß3 transmembrane complex.

Cationic membrane-proximal amino acids determine the topology of membrane proteins by interacting with anionic lipids that are restricted to the intracellular membrane leaflet. This mechanism implies that anionic lipids interfere with electrostatic interactions of membrane proteins. The integrin αIIbß3 transmembrane (TM) complex is stabilized by a membrane-proximal αIIb(Arg(995))-ß3(Asp(723)) interaction; here, we examine the influence of anionic lipids on this complex. Anionic lipids compete for αIIb(Arg(995)) contacts with ß3(Asp(723)) but paradoxically do not diminish the contribution of αIIb(Arg(995))-ß3(Asp(723)) to TM complex stability. Overall, anionic lipids in annular positions stabilize the αIIbß3 TM complex by up to 0.50 ± 0.02 kcal/mol relative to zwitterionic lipids in a headgroup structure-dependent manner. Comparatively, integrin receptor activation requires TM complex destabilization of 1.5 ± 0.2 kcal/mol, revealing a sizeable influence of lipid composition on TM complex stability. We implicate changes in lipid headgroup accessibility to small molecules (physical membrane characteristics) and specific but dynamic protein-lipid contacts in this TM helix-helix stabilization. Thus, anionic lipids in ubiquitous annular positions can benefit the stability of membrane proteins while leaving membrane-proximal electrostatic interactions intact.

Structural characterization of the regulatory domain of brain carnitine palmitoyltransferase 1.

Neurons contain a mammalian-specific isoform of the enzyme carnitine palmitoyltransferase 1 (CPT1C) that couples malonyl-CoA to ceramide levels thereby contributing to systemic energy homeostasis and feeding behavior. In contrast to CPT1A, which controls the rate-limiting step of long-chain fatty acid ß-oxidation in all tissues, the biochemical context and regulatory mechanism of CPT1C are unknown. CPT1 enzymes are comprised of an N-terminal regulatory domain and a C-terminal catalytic domain (CD) that are separated by two transmembrane helices. In CPT1A, the regulatory domain, termed N, adopts an inhibitory and non-inhibitory state, Nα and Nß, respectively, which differ in their association with the CD. To provide insight into the regulatory mechanism of CPT1C, we have determined the structure of its regulatory domain (residues Met1-Phe50) by NMR spectroscopy. In relation to CPT1A, the inhibitory Nα state was found to be structurally homologues whereas the non-inhibitory Nß state was severely destabilized, suggesting a change in overall regulation. The destabilization of Nß may contribute to the low catalytic activity of CPT1C relative to CPT1A and makes its association with the CD unlikely. In analogy to the stabilization of Nß by the CPT1A CD, non-inhibitory interactions of N of CPT1C with another protein may exist.

Construction of covalent membrane protein complexes and high-throughput selection of membrane mimics.

The association of transmembrane (TM) helices underlies membrane protein structure and folding. Structural studies of TM complexes are limited by complex stability and the often time-consuming selection of suitable membrane mimics. Here, methodology for the efficient, preparative scale construction of covalent TM complexes and the concomitant high-throughput selection of membrane mimics is introduced. For the employed integrin αIIbß3 model system, the methodology identified phospholipid bicelles, including their specific composition, as the best membrane mimic. The method facilitates structure determination by NMR spectroscopy as exemplified by the measurement of previously inaccessible residual dipolar couplings and (15)N relaxation parameters.

Isothermal Titration Calorimetry of Membrane Proteins.

The ability to quantify protein-protein interactions without adding labels to protein has made isothermal titration calorimetry (ITC) a preferred technique to study proteins in aqueous solution. Here, we describe the application of ITC to the study of protein-protein interactions in membrane mimics using the association of integrin αIIb and ß3 transmembrane domains in phospholipid bicelles as an example. A higher conceptual and experimental effort compared to water-soluble proteins is required for membrane proteins and rewarded with rare thermodynamic insight into this central class of proteins.

Insight Into Pathological Integrin αIIbß3 Activation From Safeguarding The Inactive State.

The inhibition of physiological activation pathways of the platelet adhesion receptor integrin αIIbß3 may fail to prevent fatal thrombosis, suggesting that the receptor is at risk of activation by yet an unidentified pathway. Here, we report the discovery and characterization of a structural motif that safeguards the receptor by selectively destabilizing its inactive state. At the extracellular membrane border, an overpacked αIIb(W968)-ß3(I693) contact prevents αIIb(Gly972) from optimally assembling the αIIbß3 transmembrane complex, which maintains the inactive state. This destabilization of approximately 1.0 kcal/mol could be mitigated by hydrodynamic forces but not physiological agonists, thereby identifying hydrodynamic forces as pathological activation stimulus. As reproductive life spans are not generally limited by cardiovascular disease, it appears that the evolution of the safeguard was driven by fatal, hydrodynamic force-mediated integrin αIIbß3 activation in the healthy cardiovascular system. The triggering of the safeguard solely by pathological stimuli achieves an effective increase of the free energy barrier between inactive and active receptor states without incurring an increased risk of bleeding. Thus, integrin αIIbß3 has evolved an effective way to protect receptor functional states that indicates the availability of a mechanical activation pathway when hydrodynamic forces exceed physiological margins.

Universal principles of membrane protein assembly, composition and evolution.

Structural diversity in α-helical membrane proteins (MP) arises from variations in helix-helix crossings and contacts that may bias amino acid usage. Here, we reveal systematic changes in transmembrane amino acid frequencies (f) as a function of the number of helices (n). For eukarya, breaks in f(n) trends of packing (Ala, Gly and Pro), polar, and hydrophobic residues identify different MP assembly principles for 2≤n≤7, 8≤n≤12 and n≥13. In bacteria, the first f break already occurs after n = 6 in correlation to an earlier n peak in MP size distribution and dominance of packing over polar interactions. In contrast to the later n brackets, the integration levels of helix bundles continuously increased in the first, most populous brackets indicating the formation of single structural units (domains). The larger first bracket of eukarya relates to a balance of polar and packing interactions that enlarges helix-helix combinatorial possibilities (MP diversity). Between the evolutionary old, packing and new, polar residues f anti-correlations extend over all biological taxa, broadly ordering them according to evolutionary history and allowing f estimates for the earliest forms of life. Next to evolutionary history, the amino acid composition of MP is determined by size (n), proteome diversity, and effective amino acid cost.

Membrane Anchoring of α-Helical Proteins: Role of Tryptophan.

The function of membrane proteins relies on a defined orientation of protein relative to lipid. In apparent correlation to protein anchoring, tryptophan residues are enriched in the lipid headgroup region. To characterize the thermodynamic and structural basis of this relationship in α-helical membrane proteins, we examined the role of three conserved tryptophans in the folding of the heterodimeric integrin αIIbß3 transmembrane (TM) complex in phospholipid bicelles and mammalian membranes. In the homogenous lipid environment of bicelles, tryptophan was replaceable by residues of distinct polarities. The appropriate polarity was guided by the electrostatic potential of the tryptophan surrounding, suggesting that tryptophan can complement diverse environments by adjusting the orientation of its anisotropic side chain to achieve site-specific anchoring. As a sole membrane anchor, tryptophan made a contribution of 0.4 kcal/mol to TM complex stability in bicelles. In membranes, it proved more difficult to replace tryptophan even by tyrosine, indicating a superior capacity to interact with heterogeneous lipids of biological membranes. Interestingly, at intracellular TM helix ends, where integrin activation is initiated, sequence motifs that interact with lipids via opposing polarity patterns were found to restrict TM helix orientations beyond tryptophan anchoring. In contrast to bicelles, phenylalanine became the least accepted substitute in membranes, demonstrating an increased role of the hydrophobic effect. Altogether, our study implicates a wide amphiphilic range of tryptophan, membrane complexity, and the hydrophobic effect to be important factors in tryptophan membrane anchoring.

Structural and thermodynamic basis of proline-induced transmembrane complex stabilization.

In membrane proteins, proline-mediated helix kinks are indispensable for the tight packing of transmembrane (TM) helices. However, kinks invariably affect numerous interhelical interactions, questioning the acceptance of proline substitutions and evolutionary origin of kinks. Here, we present the structural and thermodynamic basis of proline-induced integrin αIIbß3 TM complex stabilization to understand the introduction of proline kinks in membrane proteins. In phospholipid bicelles, the A711P substitution in the center of the ß3 TM helix changes the direction of adjacent helix segments to form a 35 ± 2° angle and predominantly repacks the segment in the inner membrane leaflet due to a swivel movement. This swivel repacks hydrophobic and electrostatic interhelical contacts within intracellular lipids, resulting in an overall TM complex stabilization of -0.82 ± 0.01 kcal/mol. Thus, proline substitutions can directly stabilize membrane proteins and such substitutions are proposed to follow the structural template of integrin αIIbß3(A711P).

Direct Evaluation of Protein-Lipid Contacts Reveals Protein Membrane Immersion and Isotropic Bicelle Structure.

The solvation of membrane proteins by both lipids and water makes their membrane immersion difficult to predict and the choice of a membrane mimic challenging. To characterize protein-lipid contacts and bicelle membrane mimics, we examined protein-lipid cross-relaxation of integrin αIIb and ß3(A711P) transmembrane helices in isotropic phospholipid bicelles (q = 0.5 and 0.7). Long-chain bicelle lipids dominated contacts with central helix segments, whereas both short- and long-chain lipids contacted the terminal turns of each helix in corroboration of the mixed bicelle model. The saturation transfer profiles from long-chain lipids directly established helix midpoints in the lipid bilayer. Lipid headgroups and water molecules engaged the side chains of buried serine and threonine in competition with intrahelical hydrogen bonding, illustrating that polar side chains seek the most favorable electrostatic contacts.

Mutation in CPT1C Associated With Pure Autosomal Dominant Spastic Paraplegia.

IMPORTANCE: The family of genes implicated in hereditary spastic paraplegias (HSPs) is quickly expanding, mostly owing to the widespread availability of next-generation DNA sequencing methods. Nevertheless, a genetic diagnosis remains unavailable for many patients. OBJECTIVE: To identify the genetic cause for a novel form of pure autosomal dominant HSP. DESIGN, SETTING, AND PARTICIPANTS: We examined and followed up with a family presenting to a tertiary referral center for evaluation of HSP for a decade until August 2014. Whole-exome sequencing was performed in 4 patients from the same family and was integrated with linkage analysis. Sanger sequencing was used to confirm the presence of the candidate variant in the remaining affected and unaffected members of the family and screen the additional patients with HSP. Five affected and 6 unaffected participants from a 3-generation family with pure adult-onset autosomal dominant HSP of unknown genetic origin were included. Additionally, 163 unrelated participants with pure HSP of unknown genetic cause were screened. MAIN OUTCOME AND MEASURE: Mutation in the neuronal isoform of carnitine palmitoyl-transferase (CPT1C) gene. RESULTS: We identified the nucleotide substitution c.109C>T in exon 3 of CPT1C, which determined the base substitution of an evolutionarily conserved Cys residue for an Arg in the gene product. This variant strictly cosegregated with the disease phenotype and was absent in online single-nucleotide polymorphism databases and in 712 additional exomes of control participants. We showed that CPT1C, which localizes to the endoplasmic reticulum, is expressed in motor neurons and interacts with atlastin-1, an endoplasmic reticulum protein encoded by the ATL1 gene known to be mutated in pure HSPs. The mutation, as indicated by nuclear magnetic resonance spectroscopy studies, alters the protein conformation and reduces the mean (SD) number (213.0 [46.99] vs 81.9 [14.2]; P < .01) and size (0.29 [0.01] vs 0.26 [0.01]; P < .05) of lipid droplets on overexpression in cells. We also observed a reduction of mean (SD) lipid droplets in primary cortical neurons isolated from Cpt1c-/- mice as compared with wild-type mice (1.0 [0.12] vs 0.44 [0.05]; P < .001), suggesting a dominant negative mechanism for the mutation. CONCLUSIONS AND RELEVANCE: This study expands the genetics of autosomal dominant HSP and is the first, to our knowledge, to link mutation in CPT1C with a human disease. The association of the CPT1C mutation with changes in lipid droplet biogenesis supports a role for altered lipid-mediated signal transduction in HSP pathogenesis.

Characterization of membrane protein interactions by isothermal titration calorimetry.

Understanding the structure, folding, and interaction of membrane proteins requires experimental tools to quantify the association of transmembrane (TM) helices. Here, we introduce isothermal titration calorimetry (ITC) to measure integrin αIIbß3 TM complex affinity, to study the consequences of helix-helix preorientation in lipid bilayers, and to examine protein-induced lipid reorganization. Phospholipid bicelles served as membrane mimics. The association of αIIbß3 proceeded with a free energy change of -4.61±0.04kcal/mol at bicelle conditions where the sampling of random helix-helix orientations leads to complex formation. At bicelle conditions that approach a true bilayer structure in effect, an entropy saving of >1kcal/mol was obtained from helix-helix preorientation. The magnitudes of enthalpy and entropy changes increased distinctly with bicelle dimensions, indicating long-range changes in bicelle lipid properties upon αIIbß3 TM association. NMR spectroscopy confirmed ITC affinity measurements and revealed αIIbß3 association and dissociation rates of 4500±100s(-1) and 2.1±0.1s(-1), respectively. Thus, ITC is able to provide comprehensive insight into the interaction of membrane proteins.

Sequence and membrane determinants of the random coil-helix transition of α-synuclein.

A random coil-helix transition underlies the association of the presynaptic protein α-synuclein (αS) with curved vesicle membranes to fold Asp2-Ala89 into a continuous helix. To clarify this transition, we examined αS folding cooperativity, helix nucleation and propagation in relation to membrane stabilization and leakage on diverse small unilamellar vesicles. The sequences centering on Phe4 and Tyr39 initiate lipid interactions and the Phe4 region nucleates the helix irrespective of the order of Ser9-Ala89. However, helix propagation is not the sum of individual αS-membrane interactions; it requires non-uniform but balanced sequence distributions of lipid affinities and helix flexibility. The attained helix propagation, like folding cooperativity, depends distinctly on membrane lipid composition and correlates to the degree of αS-conferred membrane stabilization. Contrary to classical coil-helix folding thermodynamics, helix propagation proceeds with a small gain in free energy relative to helix nucleation indicating that its binding enthalpy is expended to compensate a high entropic cost of reducing lipid-packing defects in the curved membrane. Non-saturating lipid conditions or rigidification of the αS helix triggers an increase in small unilamellar vesicle membrane leakage. Thus, αS folding parameters appear highly optimized and closely matched to stabilize and protect its target membrane. Aging-associated changes in lipid and αS concentrations may therefore alter synaptic plasticity and contribute to αS misfolding that culminates in fatal neurodegeneration.

A potentially common peptide target in secreted heat shock protein-90α for hypoxia-inducible factor-1α-positive tumors.

Deregulated accumulation of hypoxia-inducible factor-1α (HIF-1α) is a hallmark of many solid tumors. Directly targeting HIF-1α for therapeutics is challenging. Our finding that HIF-1α regulates secretion of heat shock protein-90α (Hsp90α) for cell migration raises the exciting possibility that targeting the secreted Hsp90α from HIF-1α-positive tumors has a better clinical outlook. Using the HIF-1α-positive and metastatic breast cancer cells MDA-MB-231, we show that down-regulation of the deregulated HIF-1α blocks Hsp90α secretion and invasion of the cells. Reintroducing an active, but not an inactive, HIF-1α into endogenous HIF-1α-depleted cells rescues both Hsp90α secretion and invasion. Inhibition of Hsp90α secretion, neutralization of secreted Hsp90α action, or removal of the cell surface LRP-1 receptor for secreted Hsp90α reduces the tumor cell invasion in vitro and lung colonization and tumor formation in nude mice. Furthermore, we localized the tumor-promoting effect to a 115-amino acid region in secreted Hsp90α called F-5. Supplementation with F-5 is sufficient to bypass the blockade of HIF-1α depletion and resumes invasion by the tumor cells under serum-free conditions. Because normal cells do not secrete Hsp90α in the absence of stress, drugs that target F-5 should be more effective and less toxic in treatment of HIF-1α-positive tumors in humans.