Amide Proton Chemical Shifts Research Articles

The conformational properties of alamethicin in ethanol studied by NMR.

Alamethicin, a peptide consisted of 20 amino acid residues, has been known to function as an antibiotic. The peptides self-associate in biological membranes, form an ion channel, and then induce cell death by leaking intracellular contents through a transmembrane pore of an ion channel. We investigated conformation and its thermal stability of alamethicin-A6 and -U6 in ethanol using proton nuclear magnetic resonance (NMR) spectroscopy; alamethicin-A6 and -U6 have the amino acid sequences of UPUAUAQUVUGLUPVUUQQO and UPUAUUQUVUGLUPVUUQQO, respectively, where U and O represent α-aminoisobutyric acid and phenylalaninol, respectively. As indicated by the under bars in the sequences, only the residue 6 differs between the alamethicins. We show that the alamethicins in ethanol form helix conformation in the region of the residues 2-11 and a non-regular conformation in the regions of the N- and C-termini, and that the helices are maintained up to 66°C at least. Conformations in the region of the residues 12-18 of the alamethicins, however, are not well identified due to the lack of NMR data. In addition, we demonstrate that the amide proton chemical shift temperature coefficients' method, which is known as an indicator for intramolecular hydrogen bonds in peptides and proteins in aqueous solutions, can be also applied to the alamethicins in ethanol. Further, we show that the conformation around the C-terminus of alamethicin-A6 is restrained by intramolecular hydrogen bonds, whereas that of alamethicin-U6 is either restrained or unrestrained by intramolecular hydrogen bonds; the alamethicin-U6 molecules having the restrained and unrestrained conformations coexist in ethanol. We discuss the two types of conformations using a model chain consisting of particles linked by rigid bonds called as the free jointed chain.

α-proton Chemical Shift Index and Amide Proton Chemical Shift Temperature Coefficient of Melittin in Methanol: Indicators for a Helix Structure and an Intra-Molecular Hydrogen Bond?

The methods of the α-proton chemical shift index (CSI) and the amide proton (NH) chemical shift temperature coefficient (Δδ/ΔT) were found experimentally by a number of studies on proton NMR chemical shifts of peptides and proteins in an aqueous solution, and have been widely accepted. They provide an insight into secondary structures and intra-molecular hydrogen bonds in peptides and proteins without complex calculation. Our question is whether the methods are applicable to helical peptides in methanol. Melittin, a peptide of 26 amino acid residues found in bee venom, has been known to consist of two helices (the residues 2-11 and 13-26) connected by a kink section (the residues 11-13). Employing the methods for melittin in methanol, we have shown that most of the CSI's for the residues 3-10 and 14-26 are - 1, and the Δδ/ΔT's for the residues 5-26 except 6 and 14 are more positive than - 6 ppb/℃. If the methods are applicable to melittin in methanol, these results indicate that helix structures are formed in the regions of the residues 3-10 and 14-26 and NH's in the residues 5-26 except 6 and 14 are involved in intra-molecular hydrogen bonds. The helical structure evaluated from the methods, hence, agrees nearly with the known helix structure. We also apply the methods to D-Pro14 melittin (synthetic melittin) and alamethicin (a peptide of 20 amino acid residues) in methanol, and show that they virtually work well.

Disorder in the Human Skp1 Structure is the Key to its Adaptability to Bind Many Different Proteins in the SCF Complex Assembly

Skp1(S-phase kinase-associated protein 1 - Homo sapiens) is an adapter protein of the SCF(Skp1-Cullin1-Fbox) complex, which links the constant components (Cul1-RBX) and the variable receptor (F-box proteins) in Ubiquitin E3 ligase. It is intriguing how Skp1 can recognise and bind to a variety of structurally different F-box proteins. For practical reasons, previous efforts have used truncated Skp1, and thus it has not been possible to track the crucial aspects of the substrate recognition process. In this background, we report the solution structure of the full-length Skp1 protein determined by NMR spectroscopy for the first time and investigate the sequence-dependent dynamics in the protein. The solution structure reveals that Skp1 has an architecture: β1-β2-H1-H2-L1–H3-L2-H4-H5-H6-H7(partially formed) and a long tail-like disordered C-terminus. Structural analysis using DALI (Distance Matrix Alignment) reveals conserved domain structure across species for Skp1. Backbone dynamics investigated using NMR relaxation suggest substantial variation in the motional timescales along the length of the protein. The loops and the C-terminal residues are highly flexible, and the (R2/R1) data suggests μs-ms timescale motions in the helices as well. Further, the dependence of amide proton chemical shift on temperature and curved profiles of their residuals indicate that the residues undergo transitions between native state and excited state. The curved profiles for several residues across the length of the protein suggest that there are native-like low-lying excited states, particularly for several C-terminal residues. Our results provide a rationale for how the protein can adapt itself, bind, and get functionally associated with other proteins in the SCF complex by utilising its flexibility and conformational sub-states.

Anomalous amide proton chemical shifts as signatures of hydrogen bonding to aromatic sidechains.

Hydrogen bonding between an amide group and the p- cloud of an aromatic ring was first identified in a protein in the 1980s. Subsequent surveys of high-resolution X-ray crystal structures found multiple instances, but their preponderance was determined to be infrequent. Hydrogen atoms participating in a hydrogen bond to the p- cloud of an aromatic ring are expected to experience an upfield chemical shift arising from a shielding ring current shift. We surveyed the Biological Magnetic Resonance Data Bank for amide hydrogens exhibiting unusual shifts as well as corroborating nuclear Overhauser effects between the amide protons and ring protons. We found evidence that Trp residues are more likely to be involved in p- hydrogen bonds than other aromatic amino acids, whereas His residues are more likely to be involved in in-plane hydrogen bonds, with a ring nitrogen acting as the hydrogen acceptor. The p- hydrogen bonds may be more abundant than previously believed. The inclusion in NMR structure refinement protocols of shift effects in amide protons from aromatic sidechains, or explicit hydrogen bond restraints between amides and aromatic rings, could improve the local accuracy of sidechain orientations in solution NMR protein structures, but their impact on global accuracy is likely be limited.

Confronting the Invisible: Assignment of Protein 1HN Chemical Shifts in Cases of Extreme Broadening.

NMR studies of intrinsically disordered proteins (IDPs) at neutral pH values are hampered by the rapid exchange of backbone amide protons with solvent. Although exchange rates can be modulated by changes in pH, interactions between IDPs that lead to phase separation sometimes only occur at neutral pH values or higher, where backbone amide-based experiments fail. Here we describe a simple NMR experiment for measuring amide proton chemical shifts in cases where 1HN spectra cannot be obtained. The approach uses a weak 1H B1 field, searching for elusive 1HN resonance frequencies that become encoded in the intensities of cross-peaks in three-dimensional 1Hα-detect spectra. Applications to the CAPRIN1 protein in both dilute- and phase-separated states highlight the utility of the method, establishing that accurate 1HN chemical shifts can be obtained even in cases where solvent hydrogen exchange rates are on the order of 1500 s-1.

Temperature dependence of NMR chemical shifts: Tracking and statistical analysis.

Isotropic chemical shifts measured by solution nuclear magnetic resonance (NMR) spectroscopy offer extensive insights into protein structure and dynamics. Temperature dependences add a valuable dimension; notably, the temperature dependences of amide proton chemical shifts are valuable probes of hydrogen bonding, temperature‐dependent loss of structure, and exchange between distinct protein conformations. Accordingly, their uses include structural analysis of both folded and disordered proteins, and determination of the effects of mutations, binding, or solution conditions on protein energetics. Fundamentally, these temperature dependences result from changes in the local magnetic environments of nuclei, but correlations with global thermodynamic parameters measured via calorimetric methods have been observed. Although the temperature dependences of amide proton and nitrogen chemical shifts are often well approximated by a linear model, deviations from linearity are also observed and may be interpreted as evidence of fast exchange between distinct conformational states. Here, we describe computational methods, accessible via the Shift‐T web server, including an automated tracking algorithm that propagates initial (single temperature) 1H—15N cross peak assignments to spectra collected over a range of temperatures. Amide proton and nitrogen temperature coefficients (slopes determined by fitting chemical shift vs. temperature data to a linear model) are subsequently calculated. Also included are methods for the detection of systematic, statistically significant deviation from linearity (curvature) in the temperature dependences of amide proton chemical shifts. The use and utility of these methods are illustrated by example, and the Shift‐T web server is freely available at http://meieringlab.uwaterloo.ca/shiftt.

On identifying low energy conformational excited states with differential ruggedness in human γS-crystallin promoting severe infantile cataracts

Transient excited states in proteins can be accurately probed from temperature dependence of amide proton (1HN) chemical shifts displaying significant curvatures. Characterizing these near-native alternative states is of high therapeutic relevance in conformational diseases wherein missense mutations promote structural instability that leads to conformational heterogeneity. Extending the structure-function paradigm from physiology to pathology, we recently reported the solution NMR structure and dynamics of a severe congenital cataract variant, G57W of human γS-crystallin (abbreviated as γS-G57W) which is resistant towards crystallization. In an endeavour to explore the functional consequences of this mutation, here we report for the first time, native state conformational ruggedness in human γS-G57W as compared to its wild-type counterpart from residue resolved nonlinear temperature dependence of backbone 1HN chemical shifts using solution NMR spectroscopy. Our calculations suggest that the simulated chemical shift curvatures are indicative of low energy excited states within 2–4 kcal mol−1 from the native state. Residues accessing alternative conformations populate the N-terminal domain of γS-G57W more than its C-terminal counterpart. Collectively, curvatures retaining native state ensemble on mild denaturation suggest that the free energy landscape in human γS-G57W at the bottom of the folding funnel is sufficiently robust and malleable against such perturbations. Overall, this critical study highlights the functional aspects of such structural malleability promoting infantile cataracts as a global health risk marker.

NMR characterization of conformational fluctuations and noncovalent interactions of SUMO protein from Drosophila melanogaster (dSmt3).

Structural heterogeneity in the native-state ensemble of dSmt3, the only small ubiquitin-like modifier (SUMO) in Drosophila melanogaster, was investigated and compared with its human homologue SUMO1. Temperature dependence of amide proton's chemical shift was studied to identify amino acids possessing alternative structural conformations in the native state. Effect of small concentration of denaturant (1M urea) on this population was also monitored to assess the ruggedness of near-native energy landscape. Owing to presence of many such amino acids, especially in the β2 -loop-α region, the native state of dSmt3 seems more flexible in comparison to SUMO1. Information about backbone dynamics in ns-ps timescale was quantified from the measurement of 15 N-relaxation experiments. Furthermore, the noncovalent interaction of dSmt3 and SUMO1 with Daxx12 (Daxx729 DPEEIIVLSDSD740 ), a [V/I]-X-[V/I]-[V/I]-based SUMO interaction motif, was characterized using Bio-layer Interferometery and NMR spectroscopy. Daxx12 fits itself in the groove formed by β2 -loop-α structural region in both dSmt3 and SUMO1, but the binding is stronger with the former. Flexibility of β2 -loop-α region in dSmt3 is suspected to assist its interaction with Daxx12. Our results highlight the role of native-state flexibility in assisting noncovalent interactions of SUMO proteins especially in organisms where a single SUMO isoform has to tackle multiple substrates single handedly.

Coupled Transmembrane Substrate Docking and Helical Unwinding in Intramembrane Proteolysis of Amyloid Precursor Protein

Intramembrane-cleaving proteases (I-CLiPs) play crucial roles in physiological and pathological processes, such as Alzheimer’s disease and cancer. However, the mechanisms of substrate recognition by I-CLiPs remain poorly understood. The aspartic I-CLiP presenilin is the catalytic subunit of the γ-secretase complex, which releases the amyloid-β peptides (Aβs) through intramembrane proteolysis of the transmembrane domain of the amyloid precursor protein (APPTM). Here we used solution NMR to probe substrate docking of APPTM to the presenilin homologs (PSHs) MCMJR1 and MAMRE50, which cleaved APPTM in the NMR tube. Chemical shift perturbation (CSP) showed juxtamembrane regions of APPTM mediate its docking to MCMJR1. Binding of the substrate to I-CLiP decreased the magnitude of amide proton chemical shifts δH at the C-terminal half of the substrate APPTM, indicating that the docking to the enzyme weakens helical hydrogen bonds and unwinds the substrate transmembrane helix around the initial ε-cleavage site. The APPTM V44M substitution linked to familial AD caused more CSP and helical unwinding around the ε-cleavage site. MAMRE50, which cleaved APPTM at a higher rate, also caused more CSP and helical unwinding in APPTM than MCMJR1. Our data suggest that docking of the substrate transmembrane helix and helical unwinding is coupled in intramembrane proteolysis and FAD mutation modifies enzyme/substrate interaction, providing novel insights into the mechanisms of I-CLiPs and AD drug discovery.

Resonance assignment of disordered protein with repetitive and overlapping sequence using combinatorial approach reveals initial structural propensities and local restrictions in the denatured state.

NMR resonance assignment of intrinsically disordered proteins poses a challenge because of the limited dispersion of amide proton chemical shifts. This becomes even more complex with the increase in the size of the system. Residue specific selective labeling/unlabeling experiments have been used to resolve the overlap, but require multiple sample preparations. Here, we demonstrate an assignment strategy requiring only a single sample of uniformly labeled (13)C,(15)N-protein. We have used a combinatorial approach, involving 3D-HNN, CC(CO)NH and 2D-MUSIC, which allowed us to assign a denatured centromeric protein Cse4 of 229 residues. Further, we show that even the less sensitive experiments, when used in an efficient manner can lead to the complete assignment of a complex system without the use of specialized probes in a relatively short time frame. The assignment of the amino acids discloses the presence of local structural propensities even in the denatured state accompanied by restricted motion in certain regions that provides insights into the early folding events of the protein.

Solution Structural Studies of GTP:Adenosylcobinamide-Phosphateguanylyl Transferase (CobY) from Methanocaldococcus jannaschii.

GTP:adenosylcobinamide-phosphate (AdoCbi-P) guanylyl transferase (CobY) is an enzyme that transfers the GMP moiety of GTP to AdoCbi yielding AdoCbi-GDP in the late steps of the assembly of Ado-cobamides in archaea. The failure of repeated attempts to crystallize ligand-free (apo) CobY prompted us to explore its 3D structure by solution NMR spectroscopy. As reported here, the solution structure has a mixed α/β fold consisting of seven β-strands and five α-helices, which is very similar to a Rossmann fold. Titration of apo-CobY with GTP resulted in large changes in amide proton chemical shifts that indicated major structural perturbations upon complex formation. However, the CobY:GTP complex as followed by 1H-15N HSQC spectra was found to be unstable over time: GTP hydrolyzed and the protein converted slowly to a species with an NMR spectrum similar to that of apo-CobY. The variant CobYG153D, whose GTP complex was studied by X-ray crystallography, yielded NMR spectra similar to those of wild-type CobY in both its apo- state and in complex with GTP. The CobYG153D:GTP complex was also found to be unstable over time.

Comparison of backbone dynamics of the type III antifreeze protein and antifreeze-like domain of human sialic acid synthase.

Antifreeze proteins (AFPs) are found in a variety of cold-adapted (psychrophilic) organisms to promote survival at subzero temperatures by binding to ice crystals and decreasing the freezing temperature of body fluids. The type III AFPs are small globular proteins that consist of one α-helix, three 3(10)-helices, and two β-strands. Sialic acids play important roles in a variety of biological functions, such as development, recognition, and cell adhesion and are synthesized by conserved enzymatic pathways that include sialic acid synthase (SAS). SAS consists of an N-terminal catalytic domain and a C-terminal antifreeze-like (AFL) domain, which is similar to the type III AFPs. Despite having very similar structures, AFL and the type III AFPs exhibit very different temperature-dependent stability and activity. In this study, we have performed backbone dynamics analyses of a type III AFP (HPLC12 isoform) and the AFL domain of human SAS (hAFL) at various temperatures. We also characterized the structural/dynamic properties of the ice-binding surfaces by analyzing the temperature gradient of the amide proton chemical shift and its correlation with chemical shift deviation from random coil. The dynamic properties of the two proteins were very different from each other. While HPLC12 was mostly rigid with a few residues exhibiting slow motions, hAFL showed fast internal motions at low temperature. Our results provide insight into the molecular basis of thermostability and structural flexibility in homologous psychrophilic HPLC12 and mesophilic hAFL proteins.

Isomeric Model Antibiotic Peptides Differing Only in Charge Placement Adopt Different Helical Conformations

The efficacy of existing antibiotics is slowly declining as species of bacteria are evolving, increasing the need to find new antibiotics. One promising opportunity lies in peptide antibiotics, which are commonly helical and cationic. Our model antibiotic peptides are composed primarily of the hydrophobic dialkylated amino acid Aib (α -aminoisobutyric acid), which imparts a strong 310-helical bias due to steric hindrance at the α-carbon. Cationic lysine residues are substituted into strategic locations in the sequence. Previous studies have reported that substitution of monoalkylated amino acids into an Aib-rich sequence can impact helical shape. We report here the effect of charge placement on peptide helical structure in two solvents: DMSO (dimethyl sulfoxide), and water. We are focusing on two octameric peptides, in which two lysine residues are placed in adjacent locations in the sequence (KK45), or one turn apart (KK36). NMR data indicates that in DMSO, KK36 adopts a canonical 310-helical structure, while the KK45 helix is kinked. However, circular dichroism spectra and NMR measurements of amide temperature coefficients for aqueous KK36 are not consistent with a canonical 310-helical conformation. We present here the complete titration of amide proton chemical shifts for KK36 and KK45 from aqueous (90:10 H2O/D2O) solution to DMSO-d6 in order to identify the residues that are undergoing local environment changes as the bulk environment is changed. Measurements of the CD spectra of both peptides aqueous solution as a function of temperature will be correlated with the amide temperature coefficient data from NMR to obtain a more complete picture of solvent-induced conformational changes. Ultimately the correlation of structure and sequence will inform future antibiotic peptide design.

Conformations of a model cyclic hexapeptide, CYIQNC: 1H-NMR and molecular dynamics studies

Solution conformation of the cyclic hexapeptide sequence, [cyclo-S-Cys-Tyr-Ile-Gln-Asn-Cys-S] (CYIQNC) – a disulfide-linked fragment of a neurohypophyseal peptide hormone oxytocin (OT) – has been investigated by high-field one-dimensional (1D) and two-dimensional (2D) NMR spectroscopic methods and compared with the results obtained from computer simulation studies. 1H-NMR results based on temperature dependence of amide proton chemical shifts and nuclear Overhauser effect indicate that peptide in solution populates different conformations, characterized by two fused β-turns. The segment Ile3-Gln4-Asn5-Cys6 yields a preferred type-III β-turn at residues 4, 5 (HB, 3HN → 6CO), while the segment Cys6, Cys1-Tyr2-Ile3 exhibits inherently weaker, flexible β-turn either of type I/II’/III/half-turn at residues 1, 2 (HB, 6HN → 3CO). The computer simulation studies using a mixed protocol of distance geometry-simulated annealing followed by constrained minimization, restrained molecular dynamics, and energy minimization showed the possibility of existence of additional conformations with the hydrogen bonds, (a) 5HN → 3CO and (b) 2HN → 6CO. These results, therefore, indicate that the additional conformations obtained from both NMR and simulation studies can also be possible to the peptide. These additional conformations might have very small population in the solution and did not show their signatures in these conditions. These findings will be helpful in designing more analogs with modifications in the cyclic moiety of OT.

A reduced dimensionality NMR pulse sequence and an efficient protocol for unambiguous assignment in intrinsically disordered proteins.

Resonance assignment in intrinsically disordered proteins poses a great challenge because of poor chemical shift dispersion in most of the nuclei that are commonly monitored. Reduced dimensionality (RD) experiments where more than one nuclei are co-evolved simultaneously along one of the time axes of a multi-dimensional NMR experiment help to resolve this problem partially, and one can conceive of different combinations of nuclei for co-evolution depending upon the magnetization transfer pathways and the desired information content in the spectrum. Here, we present a RD experiment, (4,3)D-hNCOCAnH, which uses a combination of CO and CA chemical shifts along one of the axes of the 3-dimensional spectrum, to improve spectral dispersion on one hand, and provide information on four backbone atoms of every residue-HN, N, CA and CO chemical shifts-from a single experiment, on the other. The experiment provides multiple unidirectional sequential (i → i - 1) amide (1)H correlations along different planes of the spectrum enabling easy assignment of most nuclei along the protein backbone. Occasional ambiguities that may arise due to degeneracy of amide proton chemical shifts are proposed to be resolved using the HNN experiment described previously (Panchal et al. in J Biomol NMR 20:135-147, 2001). Applications of the experiment and the assignment protocol have been demonstrated using intrinsically disordered α-synuclein (140 aa) protein.

Protein structure validation and refinement using amide proton chemical shifts derived from quantum mechanics.

We present the ProCS method for the rapid and accurate prediction of protein backbone amide proton chemical shifts - sensitive probes of the geometry of key hydrogen bonds that determine protein structure. ProCS is parameterized against quantum mechanical (QM) calculations and reproduces high level QM results obtained for a small protein with an RMSD of 0.25 ppm (r = 0.94). ProCS is interfaced with the PHAISTOS protein simulation program and is used to infer statistical protein ensembles that reflect experimentally measured amide proton chemical shift values. Such chemical shift-based structural refinements, starting from high-resolution X-ray structures of Protein G, ubiquitin, and SMN Tudor Domain, result in average chemical shifts, hydrogen bond geometries, and trans-hydrogen bond (h3 JNC') spin-spin coupling constants that are in excellent agreement with experiment. We show that the structural sensitivity of the QM-based amide proton chemical shift predictions is needed to obtain this agreement. The ProCS method thus offers a powerful new tool for refining the structures of hydrogen bonding networks to high accuracy with many potential applications such as protein flexibility in ligand binding.

Automated Fragmentation QM/MM Calculation of Amide Proton Chemical Shifts in Proteins with Explicit Solvent Model.

We have performed a density functional theory (DFT) calculation of the amide proton NMR chemical shift in proteins using a recently developed automated fragmentation quantum mechanics/molecular mechanics (AF-QM/MM) approach. Systematic investigation was carried out to examine the influence of explicit solvent molecules, cooperative hydrogen bonding effects, density functionals, size of the basis sets, and the local geometry of proteins on calculated chemical shifts. Our result demonstrates that the predicted amide proton ((1)HN) NMR chemical shift in explicit solvent shows remarkable improvement over that calculated with the implicit solvation model. The cooperative hydrogen bonding effect is also shown to improve the accuracy of (1)HN chemical shifts. Furthermore, we found that the OPBE exchange-correlation functional is the best density functional for the prediction of protein (1)HN chemical shifts among a selective set of DFT methods (namely, B3LYP, B3PW91, M062X, M06L, mPW1PW91, OB98, OPBE), and the locally dense basis set of 6-311++G**/4-31G* is shown to be sufficient for (1)HN chemical shift calculation. By taking ensemble averaging into account, (1)HN chemical shifts calculated by the AF-QM/MM approach can be used to validate the performance of various force fields. Our study underscores that the electronic polarization of protein is of critical importance to stabilizing hydrogen bonding, and the AF-QM/MM method is able to describe the local chemical environment in proteins more accurately than most widely used empirical models.

Pressure Dependence of 15N Chemical Shifts in Model Peptides Ac-Gly-Gly-X-Ala-NH2

High pressure NMR spectroscopy has developed into an important tool for studying conformational equilibria of proteins in solution. We have studied the amide proton and nitrogen chemical shifts of the 20 canonical amino acids X in the random-coil model peptide Ac-Gly-Gly-X-Ala-NH2, in a pressure range from 0.1 to 200 MPa, at a proton resonance frequency of 800 MHz. The obtained data allowed the determination of first and second order pressure coefficients with high accuracy at 283 K and pH 6.7. The mean first and second order pressure coefficients <B115N> and <B215N> for nitrogen are 2.91 ppm/GPa and −2.32 ppm/GPa2, respectively. The corresponding values <B11H> and <B21H> for the amide protons are 0.52 ppm/GPa and −0.41 ppm/GPa2. Residual dependent 1J1H15N-coupling constants are shown.

Cold-induced changes in the protein ubiquitin.

Conformational changes are essential for protein-protein and protein-ligand recognition. Here we probed changes in the structure of the protein ubiquitin at low temperatures in supercooled water using NMR spectroscopy. We demonstrate that ubiquitin is well folded down to 263 K, although slight rearrangements in the hydrophobic core occur. However, amide proton chemical shifts show non-linear temperature dependence in supercooled solution and backbone hydrogen bonds become weaker in the region that is most prone to cold-denaturation. Our data suggest that the weakening of the hydrogen bonds in the β-sheet of ubiquitin might be one of the first events that occur during cold-denaturation of ubiquitin. Interestingly, the same region is strongly involved in ubiquitin-protein complexes suggesting that this part of ubiquitin more easily adjusts to conformational changes required for complex formation.

Discovery of Marinopyrrole A (Maritoclax) as a Selective Mcl-1 Antagonist that Overcomes ABT-737 Resistance by Binding to and Targeting Mcl-1 for Proteasomal Degradation

The anti-apoptotic Bcl-2 family of proteins, including Bcl-2, Bcl-X(L) and Mcl-1, are well-validated drug targets for cancer treatment. Several small molecules have been designed to interfere with Bcl-2 and its fellow pro-survival family members. While ABT-737 and its orally active analog ABT-263 are the most potent and specific inhibitors to date that bind Bcl-2 and Bcl-X(L) with high affinity but have a much lower affinity for Mcl-1, they are not very effective as single agents in certain cancer types because of elevated levels of Mcl-1. Accordingly, compounds that specifically target Mcl-1 may overcome this resistance. In this study, we identified and characterized the natural product marinopyrrole A as a novel Mcl-1-specific inhibitor and named it maritoclax. We found that maritoclax binds to Mcl-1, but not Bcl-X(L), and is able to disrupt the interaction between Bim and Mcl-1. Moreover, maritoclax induces Mcl-1 degradation via the proteasome system, which is associated with the pro-apoptotic activity of maritoclax. Importantly, maritoclax selectively kills Mcl-1-dependent, but not Bcl-2- or Bcl-X(L)-dependent, leukemia cells and markedly enhances the efficacy of ABT-737 against hematologic malignancies, including K562, Raji, and multidrug-resistant HL60/VCR, by ∼60- to 2000-fold at 1-2 μM. Taken together, these results suggest that maritoclax represents a new class of Mcl-1 inhibitors, which antagonizes Mcl-1 and overcomes ABT-737 resistance by targeting Mcl-1 for degradation.