An isoform-selective inhibitor of tropomyosin receptor kinase A behaves as molecular glue.

The production of TrkA-selective inhibitors is considerably difficult because the kinase domains of TrkA and its isoforms TrkB/C have highly homologous amino acid sequences. Here we describe the structural basis for the acquisition of selectivity for a isoform-selective TrkA inhibitor, namely compound V1. The X-ray structure revealed that V1 acts as a molecular glue to stabilize the symmetrical dimer of the TrkA kinase domains. V1 binds to the ATP-binding site and simultaneously engages in the dimeric interface of TrkA. The region of the dimeric interface in TrkA is not conserved in TrkB/C; thus, dimer formation may be a novel mechanism for the production of selective TrkA inhibitors. The biochemical and biophysical assay results confirmed that V1 selectively inhibited TrkA and induced the dimer formation of TrkA, but not TrkB. The binding pocket at the TrkA dimer interface can be used for the production of new isoform-selective TrkA inhibitors.

The juxtamembrane region of TrkA kinase is critical for inhibitor selectivity.

Although numerous crystal structures for protein kinases have been reported, many include only the kinase domain but not the juxtamembrane (JM) region, a critical activity-controlling segment of receptor tyrosine kinases (RTKs). In this study, we determined the X-ray crystal structure of the tropomyosin receptor kinase (Trk) A selective inhibitor A1 complexed with the TrkA kinase domain and the JM region. This structure revealed that the unique inhibitor-binding pocket created by a novel JM configuration yields significant potency and high selectivity against TrkB and TrkC. Moreover, we validated the importance of the JM region for the potency of A1 using in vitro assays. The introduction of moieties that interact with the JM region will be one of the most effective strategies for producing highly selective RTK inhibitors.

Dual role of the receptor Tom20 in specificity and efficiency of protein import into mitochondria.

Mitochondria import most of their resident proteins from the cytosol, and the import receptor Tom20 of the outer-membrane translocator TOM40 complex plays an essential role in specificity of mitochondrial protein import. Here we analyzed the effects of Tom20 binding on NMR spectra of a long mitochondrial presequence and found that it contains two distinct Tom20-binding elements. In vitro import and cross-linking experiments revealed that, although the N-terminal Tom20-binding element is essential for targeting to mitochondria, the C-terminal element increases efficiency of protein import in the step prior to translocation across the inner membrane. Therefore Tom20 has a dual role in protein import into mitochondria: recognition of the targeting signal in the presequence and tethering the presequence to the TOM40 complex to increase import efficiency.

Structural basis of yeast Tim40/Mia40 as an oxidative translocator in the mitochondrial intermembrane space.

The mitochondrial intermembrane space (IMS) contains many small cysteine-bearing proteins, and their passage across the outer membrane and subsequent folding require recognition and disulfide bond transfer by an oxidative translocator Tim40/Mia40 in the inner membrane facing the IMS. Here we determined the crystal structure of the core domain of yeast Mia40 (Mia40C4) as a fusion protein with maltose-binding protein at a resolution of 3 A. The overall structure of Mia40C4 is a fruit-dish-like shape with a hydrophobic concave region, which accommodates a linker segment of the fusion protein in a helical conformation, likely mimicking a bound substrate. Replacement of the hydrophobic residues in this region resulted in growth defects and impaired assembly of a substrate protein. The Cys296-Cys298 disulfide bond is close to the hydrophobic concave region or possible substrate-binding site, so that it can mediate disulfide bond transfer to substrate proteins. These results are consistent with the growth phenotypes of Mia40 mutant cells containing Ser replacement of the conserved cysteine residues.

Evaluating the correlation of binding affinities between isothermal titration calorimetry and fragment molecular orbital method of estrogen receptor beta with diarylpropionitrile (DPN) or DPN derivatives.

Estrogen receptors (ERs) are ligand-activated transcription factors, with two subtypes ERα and ERß. The endogenous ligand of ERs is the common 17ß-estradiol, and the ligand-binding pocket of ERα and ERß is very similar. Nevertheless, some ERß-selective agonist ligands have been reported. DPN (diarylpropionitrile) is a widely used ERß-selective agonist; however, the structure of the ERß-DPN complex has not been solved. Therefore, the bound-state conformation of DPN and its enantioselectivity remain unresolved. In this report, we present the structures of the complexes of ERß with DPN or its derivatives that include a chlorine atom by the X-ray crystallography. Additionally, we measured the binding affinity between ERß and DPN or derivatives by isothermal titration calorimetry (ITC) and estimated the binding affinity by fragment molecular orbital (FMO) calculations. We also examined the correlation between the ITC data and results from the FMO calculations. FMO calculations showed that S-DPN interacts strongly with three amino acids (Glu305, Phe356, and His475) of ERß, and ITC measurements confirmed that the chlorine atom of the DPN derivatives enhances binding affinity. The enthalpy change by ITC correlated strongly with the interaction energy (total IFIEs; inter-fragment interaction energies) calculated by FMO (R = 0.870). We propose that FMO calculations are a valuable approach for enhancing enthalpy contributions in drug design, and its scope of applications includes halogen atoms such as chlorine. This study is the first quantitative comparison of thermodynamic parameters obtained from ITC measurements and FMO calculations, providing new insights for future precise drug design.

Photoaffinity Labeling of the Human A2A Adenosine Receptor and Cross-link Position Analysis by Mass Spectrometry.

Photoaffinity labeling (PAL) is widely used for the identification of ligand-binding proteins and elucidation of ligand-binding sites. PAL has also been employed for the characterization of G protein-coupled receptors (GPCRs); however, a limited number of reports has successfully identified their cross-linked amino acids. This report is the first of its kind to determine the cross-link position of the human A2A adenosine receptor by PAL with the novel diazirine-based photoaffinity probe 9.

Identification of a novel member of yeast mitochondrial Hsp70-associated motor and chaperone proteins that facilitates protein translocation across the inner membrane.

Here, we report the identification of yeast 15-kD Tim15/Zim17, a new member of mitochondrial Hsp70 (mtHsp70)-associated motor and chaperone (MMC) proteins. The 15-kD MMC protein is a peripheral inner membrane protein with a zinc-finger motif. Depletion of the 15-kD protein led to impaired import of presequence-containing proteins into the matrix in vivo and in vitro. Overexpression of the 15-kD protein rescued the functional defects of mtHsp70 in ssc1-3 cells, and a fusion protein containing the 15-kD protein physically interacts with purified mtHsp70. Tim15/Zim17 therefore cooperates with mtHsp70 to facilitate import of presequence-containing proteins into the matrix.

Structural basis for unfolding pathway-dependent stability of proteins: vectorial unfolding versus global unfolding.

Point mutations in proteins can have different effects on protein stability depending on the mechanism of unfolding. In the most interesting case of I27, the Ig-like module of the muscle protein titin, one point mutation (Y9P) yields opposite effects on protein stability during denaturant-induced "global unfolding" versus "vectorial unfolding" by mechanical pulling force or cellular unfolding systems. Here, we assessed the reason for the different effects of the Y9P mutation of I27 on the overall molecular stability and N-terminal unraveling by NMR. We found that the Y9P mutation causes a conformational change that is transmitted through beta-sheet structures to reach the central hydrophobic core in the interior and alters its accessibility to bulk solvent, which leads to destabilization of the hydrophobic core. On the other hand, the Y9P mutation causes a bend in the backbone structure, which leads to the formation of a more stable N-terminal structure probably through enhanced hydrophobic interactions.

Structural stabilities of different regions of the titin I27 domain contribute differently to unfolding upon mitochondrial protein import.

Protein import into mitochondria requires unfolding of the folded mature domain of precursor proteins. Here we compared the effects of amino-acid replacement between the core region and the N-terminal region of the titin I27 domain (the 27th Ig domain of human titin) on its import into isolated mitochondria when attached to a short presequence (pb(2)(35)). We found that several mutations in the core region around Trp34 of the I27 domain enhanced the import rates of the fusion proteins, while the N-terminal K6P mutation, which increases mechanical stability around the N-terminal region, decreases the import rate. When the K6P mutation is combined with core-destabilizing mutations, the import rates of the fusion proteins still decrease, unless a long segment is deleted. These results suggest that mutations in the core region could destabilize the transition state for unfolding from the intermediate with the detached N-terminal segment during import, leading to enhanced unfolding rates, although stabilization of the N-terminal region masks these effects. In other words, the rate-limiting step of the global unfolding upon import into mitochondria switches, depending on the balance between the stability of the N-terminal structure and the stability of the core region of the I27 domain.

Structural basis of functional cooperation of Tim15/Zim17 with yeast mitochondrial Hsp70.

Mitochondrial heat-shock protein 70 (mtHsp70) and its partner proteins drive protein import into the matrix. Tim15/Zim17/Hep1 is a mtHsp70 partner protein on the matrix side of the inner mitochondrial membrane. We determined the nuclear magnetic resonance (NMR) structure of the core domain of Tim15. On the basis of the NMR structure, we created Tim15 mutants and tested their ability to complement the functional defects of Tim15 depletion and to suppress self-aggregation of mtHsp70 in vivo. A pair of basic residues, Arg 106 and His 107, conserved Asp 111 and flexible loop 133-137, and were important (Arg 106-His 107 pair and Asp 111) or partly important (the loop 133-137) for yeast cell growth, mitochondrial protein import and the suppression of mtHsp70 aggregation. Therefore, the function of Tim15 in yeast cell growth is well correlated with its ability to suppress mtHsp70 aggregation, although it is still unknown whether inhibition of mtHsp70 aggregation is the primary function of Tim15.