Discovery of varlaxins, new aeruginosin-type inhibitors of human trypsins.

Low-molecular weight natural products display vast structural diversity and have played a key role in the development of novel therapeutics. Here we report the discovery of novel members of the aeruginosin family of natural products, which we named varlaxins. The chemical structures of varlaxins 1046A and 1022A were determined using a combination of mass spectrometry, analysis of one- and two-dimensional NMR spectra, and HPLC analysis of Marfey's derivatives. These analyses revealed that varlaxins 1046A and 1022A are composed of the following moieties: 2-O-methylglyceric acid 3-O-sulfate, isoleucine, 2-carboxy-6-hydroxyoctahydroindole (Choi), and a terminal arginine derivative. Varlaxins 1046A and 1022A differ in the cyclization of this arginine moiety. Interestingly, an unusual α-D-glucopyranose moiety derivatized with two 4-hydroxyphenylacetic acid residues was bound to Choi, a structure not previously reported for other members of the aeruginosin family. We sequenced the complete genome of Nostoc sp. UHCC 0870 and identified the putative 36 kb varlaxin biosynthetic gene cluster. Bioinformatics analysis confirmed that varlaxins belong to the aeruginosin family of natural products. Varlaxins 1046A and 1022A strongly inhibited the three human trypsin isoenzymes with IC50 of 0.62-3.6 nM and 97-230 nM, respectively, including a prometastatic trypsin-3, which is a therapeutically relevant target in several types of cancer. These results substantially broaden the genetic and chemical diversity of the aeruginosin family and provide evidence that the aeruginosin family is a source of strong inhibitors of human serine proteases.

Role of two sequence motifs of mesencephalic astrocyte-derived neurotrophic factor in its survival-promoting activity.

Mesencephalic astrocyte-derived neurotrophic factor (MANF) is a prosurvival protein that protects the cells when applied intracellularly in vitro or extracellularly in vivo. Its protective mechanisms are poorly known. Here we studied the role of two short sequence motifs within the carboxy-(C) terminal domain of MANF in its neuroprotective activity: the CKGC sequence (a CXXC motif) that could be involved in redox reactions, and the C-terminal RTDL sequence, an endoplasmic reticulum (ER) retention signal. We mutated these motifs and analyzed the antiapoptotic effect and intracellular localization of these mutants of MANF when overexpressed in cultured sympathetic or sensory neurons. As an in vivo model for studying the effect of these mutants after their extracellular application, we used the rat model of cerebral ischemia. Even though we found no evidence for oxidoreductase activity of MANF, the mutation of CXXC motif completely abolished its protective effect, showing that this motif is crucial for both MANF's intracellular and extracellular activity. The RTDL motif was not needed for the neuroprotective activity of MANF after its extracellular application in the stroke model in vivo. However, in vitro the deletion of RTDL motif inactivated MANF in the sympathetic neurons where the mutant protein localized to Golgi, but not in the sensory neurons where the mutant localized to the ER, showing that intracellular MANF protects these peripheral neurons in vitro only when localized to the ER.

Interaction of levosimendan with cardiac troponin C in the presence of cardiac troponin I peptides.

The interaction between troponin C (TnC) and troponin I (TnI) is essential for the regulation of muscle contraction. There are several binding sites for TnI on TnC that are differentially occupied depending on the phase of the contraction/relaxation cycle. TnI and TnC interact in an antiparallel fashion with each other. The C-domain of cTnC and the N-domain region of cTnI(residues 33-70) always interact under physiological conditions, whereas the interaction between regulatory regions of TnC and TnI (residues 128-166) is calcium dependent. Previously, it has been shown that levosimendan, a calcium sensitizer used as a treatment for acute heart failure, can interact with both domains of isolated cTnC. To understand which interaction is relevant for the mechanism of calcium sensitization, we used a more complete troponin model obtained by complexing cTnI(32-79) and cTnI(128-180) with calcium-saturated cTnC(CS). The cTnI peptides bound to cTnC(CS) to form a 1:1:1 complex. The interaction of levosimendan with this complex was followed by 1H-(15)N heteronuclear correlation spectroscopy. It was clear that based on chemical shift changes, cTnI(32-79) blocked the levosimendan interaction sites on the C-domain, whereas cTnI(128-180) did not compete with levosimendan for the binding site on the N-domain. Hence, the effective binding site of levosimendan on cTnC resulting in the calcium-sensitizing effect is located in the regulatory domain (N-domain).

Two simple NMR experiments for measuring dipolar couplings in asparagine and glutamine side chains.

Residual dipolar couplings are now widely used for structure determination of biological macromolecules. Until recently, the main focus has been on measurement of dipolar couplings in the protein main chain. However, with the aim of more complete protein structure, it is also essential to have information on the orientation of protein side chains. In addition, residual dipolar couplings can potentially be employed to study molecular dynamics. In this Communication, two simple NH(2) and spin-state edited experiments are presented for rapid and convenient determination of five residual dipolar couplings from (15)N, (1)H correlation spectrum in asparagine and glutamine side chains. The pulse sequences are demonstrated on two proteins, 30.4-kDa Cel6A in diluted liquid crystal phase and 18-kDa human cardiac troponin C in water.

A new approach for obtaining sequential assignment of large proteins.

A novel NMR experiment for obtaining sequential assignment of large proteins and protein complexes is described. The proposed method takes full advantage of transverse relaxation optimized spectroscopy (TROSY) and utilizes spin-state-selection to distinguish between intraresidual and sequential connectivities in the HNCA-TROSY-type correlation experiment. Thus, the intra- and interresidual cross peaks can be identified without relaying magnetization via carbonyl carbon, which relaxes very rapidly at the high magnetic fields where TROSY is most efficient. In addition, the presented method enables measurement of several scalar and residual dipolar couplings, which can potentially be used for structure determination of large proteins.

Methods for the measurement of (1)J(NCalpha) and (2)J(NCalpha) from a simplified 2D (13)C(alpha)-coupled (15)N SE-HSQC spectrum.

Two methods for the measurement of (2)J(NCalpha) and (1)J(NCalpha) in (15)N/(13)C-labeled small and medium-size proteins are described. The current approach is based on simplified (13)C(alpha)-coupled (15)N HSQC spectra, where the two (2)J(NCalpha) doublets are separated into two subspectra corresponding to the alpha and beta spin states of the residue's own alpha carbon. The displacement of the two (2)J(NCalpha) doublets between the two subspectra provides an accurate value for (1)J(NCalpha). The alpha/beta filtration is achieved by taking the sum and difference of the recorded complementary in-phase and antiphase J-coupled spectra. J-multiplication is utilized in one of the proposed methods. In this method, an additional coupling evolution period, which is incremented in concert with t(1), is included in the pulse sequence making it possible to scale the peak-to-peak separation.

Conformations of the regulatory domain of cardiac troponin C examined by residual dipolar couplings.

Conformations of the regulatory domain of cardiac troponin C (cNTnC) were studied by means of residual dipolar couplings measured from samples dissolved in dilute liquid crystals. Changes in the main chain HN residual dipolar couplings revealed a conformational change in cNTnC due to the complexation with the second binding region (amino acids 148-163) of cardiac troponin I (cTnI). Formation of the complex is accompanied with a molecular realignment in the liquid crystal. The residual dipolar couplings measured for apo-cNTnC and the complex with TnI were in agreement with the values computed from the corresponding closed and open solution structures, whereas for the calcium-loaded conformation the correlation and quality factor were only modest. Ca2+-cNTnC may be subject to conformational exchange. The data support the model that cardiac troponin C functions as a calcium-dependent open-closed switch, such as the skeletal troponin C.

Determination of backbone angle psi in proteins using a TROSY-based alpha/beta-HN(CO)CA-J experiment.

Transverse relaxation-optimized NMR experiment (TROSY) for the measurement of three-bond scalar coupling constant between (1)H(alpha)(i-1) and (15)N(i) defining the dihedral angle psi is described. The triple-spin-state-selective experiment allows measurement of (3)J(H(alpha)N) from (13)C(alpha), (15)N, and (1)H(N) correlation spectra H(2)O with minimum resonance overlap. Transverse relaxation of (13)C(alpha) spin is minimized by using spin-state-selective filtering and by acquiring a signal longer in (15)N-dimension in a manner of semi-constant-time TROSY evolution. The (3)J(H(alpha))(N) values obtained with the proposed alpha/beta-HN(CO)CA-J TROSY scheme are in good agreement with the values measured earlier from ubiquitin in D(2)O using the HCACO[N] experiment.

A set of HNCO-based experiments for measurement of residual dipolar couplings in 15N, 13C, (2H)-labeled proteins.

Several HNCO-based three-dimensional experiments are described for the measurement of 13C'(i - 1)-13Calpha(i - 1), 5N(i)-13C'(i - 1), 15N(i)-13Calpha(i), 15N(i)-13Calpha(i - 1), 1H(N)(i)-13Calpha(i), 1H(N)(i)-13Calpha(i - 1), and 13Calpha(i - 1)-13Cbeta(i - 1) scalar and dipolar couplings in 15N, 13C, (2H)-labelled protein samples. These pulse sequences produce spin-state edited spectra superficially resembling an HNCO correlation spectrum, allowing accurate and simple measurement of couplings without introducing additional spectral crowding. Scalar and dipolar couplings are measured with good sensitivity from relatively large proteins, as demonstrated with three proteins: cardiac Troponin C, calerythrin and ubiquitin. Measurement of several dipolar couplings between spin-1/2 nuclei using spin-state selective 3D HNCO spectra provides a wealth of structural information.

Transverse relaxation optimised spin-state selective NMR experiments for measurement of residual dipolar couplings.

Three transverse relaxation optimised NMR experiments (TROSY) for the measurement of scalar and dipolar couplings suitable for proteins dissolved in aqueous iso- and anisotropic solutions are described. The triple-spin-state-selective experiments yield couplings between 1HN-13Calpha, 15N-13Calpha, 1HN-13Calpha(i-1), 15N-13Calpha(i-1), 1HN-13C'(i-1), 15N-13C'(i-1) and 13C'(i-1)-13Calpha(i-1) without introducing nonessential spectral crowding compared with an ordinary two-dimensional 15N-1H correlation spectrum and without requiring explicit knowledge of carbon assignments. This set of alpha/beta-J-TROSY experiments is most useful for perdeuterated proteins in studies of structure-activity relationships by NMR to observe, in addition to epitopes for ligands, also conformational changes induced by binding of ligands.

Semi-constant-time HMSQC (SCT-HMSQC-HA) for the measurement of (3)J(H(N))(H(alpha)) couplings in (15)N-labeled proteins.

A simple method for accurately measuring (3)J(H(N))(H(alpha)) coupling constants in (15)N-labeled proteins is described. This semi-constant-time HMSQC-HA experiment combines the rapidity and convenience of the recently introduced CT-HMQC-HA scheme (Postingl and Otting, J. Biomol. NMR 12, 319-324 (1998)) with the high resolution and robustness of the HSQC experiment. The proposed method is demonstrated for the 76-residue human ubiquitin and Saccharopolyspora erythraea calerythrin (176 residues). Our results imply that the SCT-HMSQC-HA experiment is suitable also for proteins with less favorable NMR properties due to its good resolution and sensitivity.

Intensity modulated HSQC and HMQC: two simple methods to measure 3J(HNH)alpha in proteins.

Two methods for the measurement of homonuclear 3J(HNH)alpha coupling constants are described. Both HSQC- and HMQC-type experiments employ 'quantitative J-correlation', in which the coupling constant of interest is obtained from the intensity ratio of cross peaks of two spectra. The first spectrum is acquired with 3J(HNH)alpha evolution and the second with alpha-proton decoupling. The resolution of these methods in the F1-domain is not restricted.

HN(alpha/beta-COCA-J) experiment for measurement of (1)J(C'C(alpha)) couplings from two-dimensional

Anew method for measurement of one-bond (13)C'-(13)C(alpha) scalar and dipolar couplings from a two-dimensional [(15)N, (1)H] correlation spectrum is presented. The experiment is based on multiple-quantum coherence, which is created between nitrogen and carbonyl carbon for simultaneous evolution of (15)N chemical shift and coupling between (13)C' and (13)C(alpha). Optional subspectral editing is provided by the spin-state-selective filters. The residual dipolar dipolar contribution to the (13)C'-(13)C(alpha) coupling can be measured from these simplified [(15)N, (1)H]-HSQC-like spectra. In this way, without explicit knowledge of carbon assignments, conformational changes of proteins dissolved in dilute liquid crystals can be probed conveniently, e.g., in structure activity relationship by NMR studies. The method is demonstrated with human cardiac troponin C. Copyright 1999 Academic Press.

Measurement of (1)J(NC') and (2)J(H(N))(C') couplings from spin-state-selective two-dimensional correlation spectrum.

A method for the measurement of (1)J(NC') and (2)J(H(N))(C') coupling constants from a simplified two-dimensional [(15)N, (1)H] correlation spectrum is presented. The multiplet components of the (1)J(NC') doublet in the indirect dimension and (2)J(H(N))(C') in the direct dimension are separated into two subspectra by spin-state-selective filters. Thus each subspectrum contains no more peaks than the conventional [(15)N, (1)H]-HSQC spectrum. Furthermore, the method for the measurement of (1)J(NC') and (2)J(H(N))(C') is designed to exploit destructive relaxation interference (TROSY). The results are verified against the measurements of (1)J(NC') from spin-state-selective [(13)C', (1)H] correlation spectra recorded with additional sequence described here.

Measurement of homonuclear (2)J-couplings from spin-state selective double-/zero-quantum two-dimensional NMR spectra.

(1)H-detected two-dimensional double-/zero-quantum experiments are described for measurement of homonuclear (2)J(HH)-couplings of NH(2) or CH(2) groups in proteins. These experiments utilize multiple-quantum coherence for determination of the size and the absolute sign of the geminal scalar and dipolar couplings in the presence of broad lines. Spectra are simplified by gradient selection and spin-state selective filters.

J-multiplied HSQC (MJ-HSQC): a new method for measuring 3J(HNHalpha) couplings in 15N-labeled proteins.

A new method for the measurement of homonuclear 3J(HNHalpha) coupling constants in 15N-labeled small proteins is described. The method is based on a modified sensitivity enhanced HSQC experiment, where the 3J(HNHalpha) couplings are multiplied in the f1-dimension. The J-multiplication of homonuclear 3J(HNHalpha) couplings is based on simultaneous incrementation of 15N chemical shift and homonuclear coupling evolution periods. The time increment for the homonuclear coupling evolution period is chosen to be a suitable multiple (2N x t1) of the corresponding increment for 15N-shift evolution. This results in the splitting of the HSQC correlation in the f1-dimension by 2N x 3J(HNHalpha). Because the pulse sequence has good sensitivity and water suppression properties, it is particularly useful for natural abundance samples.