Solution NMR investigation on the structure and function of the isolated J-domain from Sis1: Evidence of transient inter-domain interactions in the full-length protein.

J-domain/Hsp40 proteins cooperate in aiding with folding in the cell by binding partially folded client proteins and delivering them to be folded by Hsp70. The delivery occurs concomitantly to the stimulation of the ATPase activity of Hsp70 via the N-terminally located J-domain. Although several lines of investigation have been used to study J-domain proteins, the presence of highly flexible domains (G/F- and G/M-rich) hold up obtaining a detailed full-length structure. In this work, we present the high-resolution structure of the J-domain and the N-terminal part of the G/F domain of Sis1, solved by NMR, and used chemical-shift perturbation approaches to further study the structure/function relationship of the Sis1/Hsp70 interaction. When the J-domain was compared to the full-length protein and to a G/M domain deletion mutant, an internal interaction patch formed by hydrophobic and positively charged residues (V2, D9, R27, T39, F52 and R73) was identified. Curiously, the same patch is protected by internal contacts in the full-length protein and, in combination with the loop containing the conserved HPD motif, participates in the interaction with Hsp70. Combined, these results suggest that the J-domain in the full-length Sis1 is in a transient intermediate conformation, in which its interacting patch is protected and, at the same time, also in a favorable condition to bind Hsp70, facilitating the interaction between the two proteins. Finally, 1D NMR experiments showed that the addition of ATP is followed by the disruption of the J-domain/Hsp70 complex, a necessary step for aiding the folding of the client protein.

1H, 15N and 13C resonance assignments of the J-domain of co-chaperone Sis1 from Saccharomyces cerevisiae.

Protein folding in the cell is usually aided by molecular chaperones, from which the Hsp70 (Hsp = heat shock protein) family has many important roles, such as aiding nascent folding and participating in translocation. Hsp70 has ATPase activity which is stimulated by binding to the J-domain present in co-chaperones from the Hsp40 family. Hsp40s have many functions, as for instance the binding to partially folded proteins to be delivered to Hsp70. However, the presence of the J-domain characterizes Hsp40s or, by this reason, as J-proteins. The J-domain alone can stimulate Hsp70 ATPase activity. Apparently, it also maintains the same conformation as in the whole protein although structural information on full J-proteins is still missing. This work reports the 1H, 15N and 13C resonance assignments of the J-domain of a Hsp40 from Saccharomyces cerevisiae, named Sis1. Secondary structure and order parameter prediction from chemical shifts are also reported. Altogether, the data show that Sis1 J-domain is highly structured and predominantly formed by α-helices, results that are in very good agreement with those previously reported for the crystallographic structure.

Structural and stability studies of the human mtHsp70-escort protein 1: an essential mortalin co-chaperone.

Mitochondrial Hsp70 is involved in both protein import and folding process, among other essential functions. In mammalian cells, due to its role in the malignant process, it receives the name of mortalin. Despite its importance in protein and mitochondrial homeostasis, mortalin tends to self-aggregate in vitro and in vivo, the later leads to mitochondrial biogenesis failure. Recently, a zinc-finger protein, named Hsp70-escort protein 1 (Hep1, also called Zim17/TIM15/DNLZ), was described as an essential human mitochondrial mortalin co-chaperone which avoids its self-aggregation. Here, we report structural studies of the human Hep1 (hHep1). The results indicate that hHep1 shares some structural similarities with the yeast ortholog despite the low identity and functional differences. We also observed that hHep1 oligomerizes in a concentration-dependent fashion and that the zinc ion, which is essential for hHep1 in vivo function, has an important protein-structure stabilizing effect.

Sir2-Related Protein 1 from Leishmania amazonensis is a glycosylated NAD+-dependent deacetylase.

Sirtuin proteins form a family of NAD+-dependent protein deacetylases that are considered potential drug targets against parasites. Here, we present the first characterization of a sirtuin orthologue from Leishmania amazonensis, an aetiological agent of American tegumentary leishmaniasis that has been the subject of many studies focused in the development of therapeutic approaches. The protein has high sequence identity with other Kinetoplastid Silent information regulator 2 Related Protein 1 (Sir2RP1) and was named LaSir2RP1. The gene exists as a single copy, encoding a monomeric protein (LaSir2RP1) of approximately 41 kDa that has NAD+-dependent deacetylase activity. LaSir2RP1 was immunodetected in total protein extracts, in cytoplasmic granules, and in the secreted material of both promastigotes and lesion-derived amastigotes. Analysis of both lectin­affinity purified promastigote and amastigote extracts revealed the presence of a major enriched protein of approximately 66 kDa that was recognized by an anti-LaSir2RP1 serum, suggesting that a parasite sirtuin could be glycosylated in vivo.

Analysis of molecular targets of Mycobacterium tuberculosis by analytical ultracentrifugation.

The interest in analytical ultracentrifugation (AUC) to analyze protein structural parameters and interactions has increased in the past decades as a result of several developments on new generation instrumentation and data analysis tools. In this article, we review AUC principles and applications to study proteins, emphasizing molecular targets of Mycobacterium tuberculosis.

Human hnRNP Q re-localizes to cytoplasmic granules upon PMA, thapsigargin, arsenite and heat-shock treatments.

Eukaryotic gene expression is regulated on different levels ranging from pre-mRNA processing to translation. One of the most characterized families of RNA-binding proteins is the group of hnRNPs: heterogenous nuclear ribonucleoproteins. Members of this protein family play important roles in gene expression control and mRNAs metabolism. In the cytoplasm, several hnRNPs proteins are involved in RNA-related processes and they can be frequently found in two specialized structures, known as GW-bodies (GWbs), previously known as processing bodies: PBs, and stress granules, which may be formed in response to specific stimuli. GWbs have been early reported to be involved in the mRNA decay process, acting as a site of mRNA degradation. In a similar way, stress granules (SGs) have been described as cytoplasmic aggregates, which contain accumulated mRNAs in cells under stress conditions and present reduced or inhibited translation. Here, we characterized the hnRNP Q localization after different stress conditions. hnRNP Q is a predominantly nuclear protein that exhibits a modular organization and several RNA-related functions. Our data suggest that the nuclear localization of hnRNP Q might be modified after different treatments, such as: PMA, thapsigargin, arsenite and heat shock. Under different stress conditions, hnRNP Q can fully co-localize with the endoplasmatic reticulum specific chaperone, BiP. However, under stress, this protein only co-localizes partially with the proteins: GW182-GWbs marker protein and TIA-1 stress granule component.

DNA and heparin chaperone the refolding of purified recombinant replication protein A subunit 1 from Leishmania amazonensis.

Replication protein A (RPA) is a single-stranded DNA-binding protein that has been implicated in DNA metabolism and telomere maintenance. Subunit 1 of RPA from Leishmania amazonensis (LaRPA-1) has previously been affinity-purified on a column containing a G-rich telomeric DNA. LaRPA-1 binds and co-localizes with parasite telomeres in vivo. Here we describe the purification and characterization of native recombinant LaRPA-1 (rLaRPA-1). The protein was initially re-solubilized from inclusion bodies by using urea. After dialysis, rLaRPA-1 was soluble but contaminated with DNA, which was removed by an anion-exchange chromatography of the protein solubilized in urea. However, rLaRPA-1 precipitated after dialysis to remove urea. To investigate whether the contaminating DNA was involved in chaperoning the refolding of rLaRPA-1, salmon sperm DNA or heparin was added to the solution before dialysis. The addition of either of these substances prevented the precipitation of rLaRPA-1. The resulting rLaRPA-1 was soluble, correctly folded, and able to bind telomeric DNA. This is the first report showing the characterization of rLaRPA1 and of the importance of additives in chaperoning the refolding of this protein. The availability of rLaRPA-1 should be helpful in assessing the importance of this protein as a potential drug target.

On the quaternary structure of a C-type lectin from Bothrops jararacussu venom--BJ-32 (BjcuL).

BJ-32 (also known as BjcuL) is a C-type lectin from the venom of Bothrops jararacussu with specificity for beta-galactosides and a remarkable ability to agglutinate several species of trypanosomatids. Our objective was to study the oligomerization state of native BJ-32 by using different biophysical and computational methods. Small-angle X-ray light scattering (SAXS) experiments disclosed a compact, globular protein with a radius of gyration of 36.72+/-0.04A and molecular weight calculated as 147.5+/-2.0kDa. From analytical ultracentrifugation analysis, it was determined that the BJ-32 sedimentation profile fits nicely to a decamer model. The analysis of the intrinsic emitted fluorescence spectra for BJ-32 solutions indicated that association of subunits in the decamer is accompanied by changes in the environment of Tryptophan residues. Both ab initio and comparative models of BJ-32 supported the resemblance of the decamer in the crystallographic structure from a close homologue, the rattlesnake venom lectin (RSL) from Crotalus atrox.

Leishmania replication protein A-1 binds in vivo single-stranded telomeric DNA.

Replication protein A (RPA) is a highly conserved heterotrimeric single-stranded DNA-binding protein involved in different events of DNA metabolism. In yeast, subunits 1 (RPA-1) and 2 (RPA-2) work also as telomerase recruiters and, in humans, the complex unfolds G-quartet structures formed by the 3' G-rich telomeric strand. In most eukaryotes, RPA-1 and RPA-2 bind DNA using multiple OB fold domains. In trypanosomatids, including Leishmania, RPA-1 has a canonical OB fold and a truncated RFA-1 structural domain. In Leishmania amazonensis, RPA-1 alone can form a complex in vitro with the telomeric G-rich strand. In this work, we show that LaRPA-1 is a nuclear protein that associates in vivo with Leishmania telomeres. We mapped the boundaries of the OB fold DNA-binding domain using deletion mutants. Since Leishmania and other trypanosomatids lack homologues of known telomere end binding proteins, our results raise questions about the function of RPA-1 in parasite telomeres.

LaRbp38: a Leishmania amazonensis protein that binds nuclear and kinetoplast DNAs.

Leishmania amazonensis causes a wide spectrum of leishmaniasis. There are no vaccines or adequate treatment for leishmaniasis, therefore there is considerable interest in the identification of new targets for anti-leishmania drugs. The central role of telomere-binding proteins in cell maintenance makes these proteins potential targets for new drugs. In this work, we used a combination of purification chromatographies to screen L. amazonensis proteins for molecules capable of binding double-stranded telomeric DNA. This approach resulted in the purification of a 38kDa polypeptide that was identified by mass spectrometry as Rbp38, a trypanosomatid protein previously shown to stabilize mitochondrial RNA and to associate with nuclear and kinetoplast DNAs. Western blotting and supershift assays confirmed the identity of the protein as LaRbp38. Competition and chromatin immunoprecipitation assays confirmed that LaRbp38 interacted with kinetoplast and nuclear DNAs in vivo and suggested that LaRbp38 may have dual cellular localization and more than one function.

Further ultrastructural characterization of the intranuclear ring-shaped bodies of the plant Lacandonia schismatica.

Ring-shaped bodies are found in the nucleus of Lacandonia schismatica, a rare plant with the sexual organs inverted. They are 0.5-microm-diameter structures that present an electron-dense external ring surrounding a central core. Ultrastructural studies indicate that these bodies contain RNA. The external ring is labeled with antibodies against small nuclear ribonucleoproteins, suggesting that they may be involved in pre-mRNA metabolism. In the present work we further characterized these intranuclear ring-shaped structures by serial-sectioning analysis. Moreover, we tested the presence of additional molecular elements related to pre-mRNA metabolism, such as SR proteins and poly(A)(+) RNA, using immunoelectron microscopy and ultrastructural in situ hybridization. Our results show that these nuclear bodies are spherical. They contain SR proteins involved in splicing and postsplicing events and little to no poly(A)(+) RNA. We also found similar nuclear bodies in other plant and animal species. Therefore, ring-shaped bodies in L. schismatica are spherical, highly compartmentalized nuclear structures that may be involved in pre-mRNA metabolism.

Classification of non-unions of the proximal humerus.

A classification of non-unions of the proximal humerus is proposed based on a group of 21 cases. Suggestions for treatment are given.

Putative interhelix ion pairs involved in the stability of myoglobin.

An earlier theoretical study predicted that specific ion pair interactions between neighboring helices should be important in stabilizing myoglobin. To measure these interactions in sperm whale myoglobin, single mutations were made to disrupt them. To obtain reliable DeltaG values, conditions were found in which the urea induced unfolding of holomyoglobin is reversible and two-state. The cyanomet form of myoglobin satisfies this condition at pH 5, 25 degrees C. The unfolding curves monitored by far-UV CD and Soret absorbance are superimposable and reversible. None of the putative ion pairs studied here makes a large contribution to the stability of native myoglobin. The protein stability does decrease somewhat between 0 and 0.1 M NaCl, however, indicating that electrostatic interactions contribute favorably to myoglobin stability at pH 5.0. A previous mutational study indicated that the net positive charge of the A[B]GH subdomain of myoglobin is an important factor affecting the stability of the pH 4 folding intermediate and potential ion pairs within the subdomain do not contribute significantly to its stability. One of the assumptions made in that study is tested here: replacement of either positively or negatively charged residues outside the A[B]GH subdomain has no significant effect on the stability of the pH 4 molten globule.

Mapping subdomains in the C-terminal region of troponin I involved in its binding to troponin C and to thin filament.

Troponin I (TnI) is the inhibitory component of troponin, the ternary complex that regulates skeletal and cardiac muscle contraction. Previous work showed that the C-terminal region of TnI, when linked to the "inhibitory region" (residues 98-116), possesses the major regulatory functions of the molecule (Farah, C. S., Miyamoto, C. A., Ramos, C. H. I., Silva, A. C. R., Quaggio, R. B., Fujimori, K., Smillie, L. B., and Reinach, F. C. (1994) J. Biol. Chem. 269, 5230-5240). To investigate these functions in more detail, serial deletion mutants of the C-terminal region of TnI were constructed. These experiments showed that longer C-terminal deletions result in lower inhibition of the actomyosin ATPase activity and weaken the interaction with the N-terminal domain of troponin C (TnC), consistent with the antiparallel model for the interaction between these two proteins. The conclusion is that the whole C-terminal region of TnI is necessary for its full regulatory activity. The region between residues 137 and 144, which was shown to have homology with residues 108-115 in the inhibitory region (Farah, C. S., and Reinach, F. C. (1995) FASEB J. 9, 755-767), is involved in the binding to TnC. The region between residues 98 and 129 is involved in modulating the affinity of TnC for calcium. The C-terminal residues 166-182 are involved in the binding of TnI to thin filament. A model for the function of TnI is discussed.

Specificity of native-like interhelical hydrophobic contacts in the apomyoglobin intermediate.

On exposure to mildly acidic conditions, apomyoglobin forms a partially folded intermediate, I. The A, B, G, and H helices are significantly structured in this equilibrium intermediate, whereas the remainder of the protein is largely unfolded. We report here the effects of mutations at helix pairing sites on the stability of I in three classes of mutants that: (i) truncate hydrophobic side chains in native helix packing sites, (ii) truncate hydrophobic side chains not involved in interhelical contacts, and (iii) extend hydrophobic side chains at residues not involved in interhelical contacts. Class I mutants significantly decrease the stability and cooperativity of folding of the intermediate. Class II and III mutants show smaller effects on stability and have little effect on cooperativity. Qualitatively similar results to those found in I were obtained for all three classes of mutants in native myoglobin (N), demonstrating that hydrophobic burial is fairly specific to native helix packing sites in I as well as in N. These results suggest that hydrophobic burial along native-like interhelical contacts is important for the formation of the cooperatively folded intermediate.

The effect of regulatory Ca2+ on the in situ structures of troponin C and troponin I: a neutron scattering study.

The effects of regulatory amounts of Ca2+ on the in situ structures of troponin C (TnC) and troponin I (TnI) in whole troponin have been investigated by neutron scattering. In separate difference experiments, 97% deuterated TnC and TnI within whole troponin were studied +/-Ca2+ in 41.6% 2H2O buffers in which protonated subunits were rendered "invisible". We found that the radius of gyration (Rg) of TnI decreased by approximately 10% upon addition of regulatory Ca2+ indicating that it was significantly more compact in the presence of Ca2+. The apparent cross-sectional radius of gyration (Rc) of TnI increased by about 9% when regulatory Ca2+ was bound to TnC. Modeling studies showed that the high-Q scattering patterns of TnI could be fit by a TnI which consisted of two subdomains: one, a highly oblate ellipsoid of revolution containing about 65% of the mass and the other, a highly prolate ellipsoid of revolution consisting of about 35% of the mass. No other fits could be found with this class of models. Best fits were achieved when the axes of revolution of these ellipsoids were steeply inclined with respect to each other. Ca2+ addition decreased the center of mass separation by about 1.5 nm. The Rg of TnI, its high-Q scattering pattern, and the resultant structure were different from previous results on neutron scattering by TnI in the (+Ca2+) TnC.TnI complex. The Rg of TnC indicated that it was elongate in situ. The Rg of TnC was not sensitive to the Ca2+ occupancy of its regulatory sites. However, Rc increased upon Ca2+ addition in concert with expectations from NMR and crystallography of isolated TnC. The present observations indicate that TnI acts like a molecular switch which is controlled by smaller Ca2+-induced changes in TnC.

Kinetic mechanism of the inhibition of human urinary kallikrein by basic pancreatic trypsin inhibitor.

Hydrolysis of D-valyl-L-leucyl-L-arginine p-nitroanilide (D-Val-Leu-Arg-Nan) at five different concentrations (10-20 microM) by human urinary kallikrein was studied in the absence and in the presence of increasing concentrations of basic pancreatic trypsin inhibitor (BPTI) (1.35-9.15 nM). The data indicate that the inhibition of human urinary kallikrein by BPTI is not a simple competitive inhibition as reported by others, but that it is a competitive inhibition of the parabolic type, with two inhibitor molecules binding to one enzyme molecule, with the formation of a ternary enzymatic complex. Statistical analysis of the experimental data supports the kinetic model proposed. The calculated values of the constants Ki and Kii were 16.20 nM and 1.10 nM, respectively. It is noteworthy that the Kii < Ki, i.e., the second BPTI molecule binds to the enzyme with a larger affinity suggesting that this second binding site was probably created or positively modulated as a consequence of the binding of the first BPTI molecule.

Kinetic mechanism of the inhibition of human urinary kallikrein by basic pancreatic trypsin inhibitor

Hydrolysis of D-valvyl-L-leucyl-L-arginine p-nitroanilide (D-Val-Leu-Arg-Nan) at five different concentration (10-20µM) by human urinary kallikrein was studied in the absence and in the presence of increasing concentrations of basic pancreatic trypsin inhibitor (BPTI) (1.35-9.15nM). The data indicate that the inhbition of human urinary kallikrein by BPTI is not a simple competitive inhibition as reported by others, but that it is a competitive inhibition of the parabolic type, with two inhibitor molecules binding to one enzyme molecule, with the formetion of a ternary enzymatic complex. Statistical analysis of the experimental data supports the kinetic model proposed. The calculated values of the constants Ki and Kii were 16.20 nM and 1.10 nM, repectively. It is noteworthy that the Kii < Ki, i.e., the second BPTI molecule binds to the enzyme with a larger affinity suggesting that this second binding site was probably created or positively modulated as a consequence of the binding of the first BPTI molecule

Structural and regulatory functions of the NH2- and COOH-terminal regions of skeletal muscle troponin I.

Calcium binding to regulatory sites located in the NH2-terminal domain of troponin C (TnC) induces a conformational change that blocks the inhibitory action of troponin I (TnI) and triggers muscle contraction. We used deletion mutants of TnI in conjunction with a series of TnC mutants to understand the structural and functional relationship between different TnI regions and TnC domains. Our results indicate that TnI is organized into structural and regulatory regions which interact in an antiparallel fashion with the corresponding structural and regulatory regions of TnC. Functional studies show that the COOH-terminal region of TnI, when linked to the inhibitory region (TnI103-182) can regulate actomyosin ATPase. A TnI lacking the first 57 amino acids (TnId57) has been shown to have similar properties (Sheng, Z., Pan, B.-S., Miller, T. E., and Potter, J. D. (1992) J. Biol. Chem. 267, 25407-25413). Regulation was not observed with the COOH-terminal region alone (TnI120-182), with the NH2-terminal region alone (TnI1-98), or with the NH2-terminal linked to the inhibitory region (TnI1-116). Binding studies show that the NH2-terminal region of TnI interacts with the COOH-terminal domain of TnC in the presence of Ca2+ or Mg2+ and that the inhibitory plus COOH-terminal region of TnI (TnI103-182) interacts with the NH2-terminal domain of TnC in a Ca(2+)-dependent manner. Based on these results we propose a model for the Ca(2+)-induced conformational change. In our model the NH2-terminal domain of TnI is anchored strongly to the COOH-terminal domain of TnC in the absence and presence of Ca2+ while the inhibitory and COOH-terminal regions of TnI switch between actin-tropomyosin in the absence of Ca2+ to binding sites in both NH2- and COOH-terminal domains of TnC in the presence of Ca2+.