PubMed Health⌕ Search

Biomedical subjects

T A Holak

Publications and source records attributed to T A Holak.

At least 19 recordsLinked to original sources
PubMed2001

The interaction of insulin-like growth factor-I with the N-terminal domain of IGFBP-5.

Insulin-like growth factors (IGFs) are key regulators of cell proliferation, differentiation and transformation, and are thus pivotal in cancer, especially breast, prostate and colon neoplasms. They are also important in many neurological and bone disorders. Their potent mitogenic and anti-apoptotic actions depend primarily on their availability to bind to the cell surface IGF-I receptor. In circulation and interstitial fluids, IGFs are largely unavailable as they are tightly associated with IGF-binding proteins (IGFBPs) and are released after IGFBP proteolysis. Here we report the 2.1 A crystal structure of the complex of IGF-I bound to the N-terminal IGF-binding domain of IGFBP-5 (mini-IGFBP-5), a prototype interaction for all N-terminal domains of the IGFBP family. The principal interactions in the complex comprise interlaced hydrophobic side chains that protrude from both IGF-I and the IGFBP-5 fragment and a surrounding network of polar interactions. A solvent-exposed hydrophobic patch is located on the IGF-I pole opposite to the mini-IGFBP-5 binding region and marks the IGF-I receptor binding site.

Amino Acid Sequence↗
PubMed2001

The extracellular human melanoma inhibitory activity (MIA) protein adopts an SH3 domain-like fold.

Melanoma inhibitory activity (MIA) protein is a clinically valuable marker in patients with malignant melanoma, as enhanced values diagnose metastatic melanoma stages III and IV. Here we show that the recombinant human MIA adopts an SH3 domain-like fold in solution, with two perpendicular, antiparallel, three- and five-stranded beta-sheets. In contrast to known structures with the SH3 domain fold, MIA is a single-domain protein, and contains an additional antiparallel beta-sheet and two disulfide bonds. MIA is also the first extracellular protein found to have the SH3 domain-like fold. Furthermore, we show that MIA interacts with fibronectin and that the peptide ligands identified for MIA exhibit a matching sequence to type III human fibronectin repeats, especially to FN14, which is close to an integrin alpha4beta1 binding site. The present study, therefore, may explain the role of MIA in metastasis in vivo, and supports a model in which the binding of human MIA to type III repeats of fibronectin competes with integrin binding, thus detaching cells from the extracellular matrix.

Amino Acid Sequence↗
PubMed2001

Chalcone derivatives antagonize interactions between the human oncoprotein MDM2 and p53.

The oncoprotein MDM2 inhibits the tumor suppressor protein p53 by binding to the p53 transactivation domain. The p53 gene is inactivated in many human tumors either by mutations or by binding to oncogenic proteins. In some tumors, such as soft tissue sarcomas, overexpression of MDM2 inactivates an otherwise intact p53, disabling the genome integrity checkpoint and allowing cell cycle progression of defective cells. Disruption of the MDM2/p53 interaction leads to increased p53 levels and restored p53 transcriptional activity, indicating restoration of the genome integrity check and therapeutic potential for MDM2/p53 binding antagonists. Here, we show by multidimensional NMR spectroscopy that chalcones (1,3-diphenyl-2-propen-1-ones) are MDM2 inhibitors that bind to a subsite of the p53 binding cleft of human MDM2. Biochemical experiments showed that these compounds can disrupt the MDM2/p53 protein complex, releasing p53 from both the p53/MDM2 and DNA-bound p53/MDM2 complexes. These results thus offer a starting basis for structure-based drug design of cancer therapeutics.

Antineoplastic Agents↗
PubMed2001

Analytical solution to the Lipari-Szabo model based on the reduced spectral density approximation offers a novel protocol for extracting motional parameters.

An analytical solution to the Lipari-Szabo model is derived for isotropic overall tumbling. The parameters of the original Lipari-Szabo model, the order parameter S2 and the effective internal correlation time tau(e), are calculated from two values of the spectral density function. If additionally the spectral density value J(0) is known, the exchange contribution R(ex) term can also be determined. The overall tumbling time tau(c) must be determined in advance, for example, from T1/T2 ratios. The required spectral density values are obtained by reduced spectral density mapping from T1, T2, and NOE measurements. Our computer simulations show that the reduced spectral density mapping is a very good approximation in almost all cases in which the Lipari-Szabo model is applicable. The robustness of the analytical formula to experimental errors is also investigated by extensive computer simulations and is found to be similar to that of the fitting procedures. The derived formulas were applied to the experimental 15N relaxation data of ubiquitin. Our results agree well with the published parameter values of S2 and tau(e), which were obtained from standard fitting procedures. The analytical approach to extract parameters of molecular motions may be more robust than standard analyses and provides a safeguard against spurious fitting results, especially for determining the exchange contribution R(ex).

Algorithms↗
PubMed2000

Solution structure of PCP, a prototype for the peptidyl carrier domains of modular peptide synthetases.

BACKGROUND: Nonribosomal peptide synthetases (NRPSs) are large modular enzymes responsible for the synthesis of a variety of microbial bioactive peptides. They consist of modules that each recognise and incorporate one specific amino acid into the peptide product. A module comprises several domains, which carry out the individual reaction steps. After activation by the adenylation domain, the amino acid substrate is covalently tethered to a 4'-phosphopantetheinyl cofactor of a peptidyl carrier domain (PCP) that passes the substrate to the reaction centres of the consecutive domains. RESULTS: The solution structure of PCP, a distinct peptidyl carrier protein derived from the equivalent domain of an NRPS, was solved using NMR techniques. PCP is a distorted four-helix bundle with an extended loop between the first two helices. Its overall fold resembles the topology of acyl carrier proteins (ACPs) from Escherichia coli fatty acid synthase and actinorhodin polyketide synthase from Streptomyces coelicolor; however, the surface polarity and the length and relative alignment of the helices are different. The conserved serine, which is the cofactor-binding site, has the same location as in the ACPs and is situated within a stretch of seven flexible residues. CONCLUSIONS: The structure of PCP reflects its character as a protein domain. The fold is well defined between residues 8 and 82 and the structural core of the PCP domain can now be defined as a region spanning 37 amino acids in both directions from the conserved serine. The flexibility of the post-translationally modified site might have implications for interactions with the cooperating proteins or NRPS domains.

Amino Acid Sequence↗
PubMed2000

Separation of anisotropy and exchange broadening using (15)N CSA-(15)N-(1)H dipole-dipole relaxation cross-correlation experiments.

Based on the measurement of cross-correlation rates between (15)N CSA and (15)N-(1)H dipole-dipole relaxation we propose a procedure for separating exchange contributions to transverse relaxation rates (R(2) = 1/T(2)) from effects caused by anisotropic rotational diffusion of the protein molecule. This approach determines the influence of anisotropy and chemical exchange processes independently and therefore circumvents difficulties associated with the currently standard use of T(1)/T(2) ratios to determine the rotational diffusion tensor. We find from computer simulations that, in the presence of even small amounts of internal flexibility, fitting T(1)/T(2) ratios tends to underestimate the anisotropy of overall tumbling. An additional problem exists when the N-H bond vector directions are not distributed homogeneously over the surface of a unit sphere, such as in helix bundles or beta-sheets. Such a case was found in segment 4 of the gelation factor (ABP 120), an F-actin cross-linking protein, in which the diffusion tensor cannot be calculated from T(1)/T(2) ratios. The (15)N CSA tensor of the residues for this beta-sheet protein was found to vary even within secondary structure elements. The use of a common value for the whole protein molecule therefore might be an oversimplification. Using our approach it is immediately apparent that no exchange broadening exists for segment 4 although strongly reduced T(2) relaxation times for several residues could be mistaken as indications for exchange processes.

Actins↗
PubMed2000

Structure of a novel leech carboxypeptidase inhibitor determined free in solution and in complex with human carboxypeptidase A2.

Leech carboxypeptidase inhibitor (LCI) is a novel protein inhibitor present in the medicinal leech Hirudo medicinalis. The structures of LCI free and bound to carboxypeptidase A2 (CPA2)have been determined by NMR and X-ray crystallography, respectively. The LCI structure defines a new protein motif that comprises a five-stranded antiparallel beta-sheet and one short alpha-helix. This structure is preserved in the complex with human CPA2 in the X-ray structure, where the contact regions between the inhibitor and the protease are defined. The C-terminal tail of LCI becomes rigid upon binding the protease as shown in the NMR relaxation studies, and it interacts with the carboxypeptidase in a substrate-like manner. The homology between the C-terminal tails of LCI and the potato carboxypeptidase inhibitor represents a striking example of convergent evolution dictated by the target protease. These new structures are of biotechnological interest since they could elucidate the control mechanism of metallo-carboxypeptidases and could be used as lead compounds for the search of fibrinolytic drugs.

Amino Acid Sequence↗
PubMed1999

Structure of the active domain of the herpes simplex virus protein ICP47 in water/sodium dodecyl sulfate solution determined by nuclear magnetic resonance spectroscopy.

ICP47 encoded by herpes simplex virus (HSV) is a key factor in the evasion of cellular immune response against HSV-infected cells. By specific inhibition of the transporter associated with antigen processing (TAP), ICP47 prevents peptide transport into the endoplasmic reticulum and subsequent loading of major histocompatibility complex (MHC) class I molecules. Amino acid residues 3-34 have been identified as the active domain. This domain appeared to be unstructured in aqueous solution, whereas after binding to membranes an alpha-helical conformation was observed. Here, we have analyzed the structure of ICP47(2-34) in a lipidlike environment by nuclear magnetic resonance (NMR) spectroscopy. In micellar solution of deuterated sodium dodecyl sulfate, the viral TAP inhibitor adopts an ordered structure. There are two helical regions extending from residues 4 to 15 and from residues 22 to 32. Arg-16 is found on the C-terminus of the first helix, and Gly-33 serves as a terminator of the second helix. A loop between residues 17 and 21 is also evident in the structure. The relative orientation of the helices toward each other, however, could not be determined due to the paucity of NOEs from residues 18-21.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1999

Molecular architecture of the rod domain of the Dictyostelium gelation factor (ABP120).

The Dictyostelium discoideum gelation factor is a two-chain actin-cross-linking protein that, in addition to an N-terminal actin-binding domain, has a rod domain constructed from six tandem repeats of a 100-residue motif that has an immunoglobulin fold. To define the architecture of the rod domain of gelation factor, we have expressed in E. coli a series of constructs corresponding to different numbers of gelation factor rod repeats and have characterised them by chemical crosslinking, ultracentrifugation, column chromatography, matrix-assisted laser desorption ionisation (MALDI) mass spectrometry and NMR spectroscopy. Fragments corresponding to repeats 1-6 and 5-6 dimerise, whereas repeats 1-5 and single repeats 3 and 4 are monomeric. Repeat 6 interacts weakly and was present as monomer and dimer when analysed by analytical ultracentrifugation. Proteolytic digestion of rod5-6 resulted in the generation of two polypeptides that roughly corresponded to rod5 and part of rod6. None of these polypeptides formed dimers after chemical crosslinking. Stable dimerisation therefore appears to require repeats 5 and 6. Based on these data a model of gelation factor architecture is presented. We suggest an arrangement of the chains where only the carboxy-terminal repeats interact as was observed for filamin/ABP280, the mammalian homologue of gelation factor.

Animals↗
PubMed1999

The domains of protein S from Myxococcus xanthus: structure, stability and interactions.

Protein S from Myxococcus xanthus is a member of the beta gamma-crystallin superfamily. Its N and C-terminal domains (NPS and CPS, respectively) show a high degree of structural similarity and possess the capacity to bind two calcium ions per domain. For NPS, their positions were determined by X-ray diffraction at 1.8 A resolution, making use of molecular replacement with the NMR structure as search model. The overall topology of NPS is found to be practically the same as in complete protein S. In natural protein S, the domains fold independently, with a significant increase in stability and cooperativity of folding in the presence of Ca2+. The recombinant isolated domains are stable monomers which do not show any tendency to combine to "nicked" full-length protein S. In order to investigate the stability and folding of natural protein S and its isolated domains, spectroscopic techniques were applied, measuring the reversible urea and temperature-induced unfolding transitions at varying pH. The increment of Ca2+ to the free energy of stabilization amounts to -10 and -5 kJ/mol for NPS and CPS, respectively. For both NPS and CPS, in the absence and in the presence of 3 mM CaCl2, the two-state model is valid. Comparing DeltaGU-->N for CPS (-21 kJ/mol at pH 7, liganded with Ca2+) with its increment in the intact two-domain protein, the stability of the isolated domain turns out to be decreased in a pH-dependent manner. In contrast, the stability of Ca2+-loaded NPS (DeltaGU-->N=-31 kJ/mol, pH 7) is nearly unchanged down to pH 2 where Ca2+ is released (DeltaGU-->N=-26 kJ/mol, pH 2). In intact protein S, the N-terminal domain is destabilized relative to NPS. Evidently, apart from Ca2+ binding, well-defined domain interactions contribute significantly to the overall stability of intact protein S.

Bacterial Proteins↗
PubMed1998

Structure of the IGF-binding domain of the insulin-like growth factor-binding protein-5 (IGFBP-5): implications for IGF and IGF-I receptor interactions.

Binding proteins for insulin-like growth factors (IGFs) IGF-I and IGF-II, known as IGFBPs, control the distribution, function and activity of IGFs in various cell tissues and body fluids. Insulin-like growth factor-binding protein-5 (IGFBP-5) is known to modulate the stimulatory effects of IGFs and is the major IGF-binding protein in bone tissue. We have expressed two N-terminal fragments of IGFBP-5 in Escherichia coli; the first encodes the N-terminal domain of the protein (residues 1-104) and the second, mini-IGFBP-5, comprises residues Ala40 to Ile92. We show that the entire IGFBP-5 protein contains only one high-affinity binding site for IGFs, located in mini-IGFBP-5. The solution structure of mini-IGFBP-5, determined by nuclear magnetic resonance spectroscopy, discloses a rigid, globular structure that consists of a centrally located three-stranded anti-parallel beta-sheet. Its scaffold is stabilized further by two inside packed disulfide bridges. The binding to IGFs, which is in the nanomolar range, involves conserved Leu and Val residues localized in a hydrophobic patch on the surface of the IGFBP-5 protein. Remarkably, the IGF-I receptor binding assays of IGFBP-5 showed that IGFBP-5 inhibits the binding of IGFs to the IGF-I receptor, resulting in reduction of receptor stimulation and autophosphorylation. Compared with the full-length IGFBP-5, the smaller N-terminal fragments were less efficient inhibitors of the IGF-I receptor binding of IGFs.

3T3 Cells↗
PubMed1998

Structure of human cyclin-dependent kinase inhibitor p19INK4d: comparison to known ankyrin-repeat-containing structures and implications for the dysfunction of tumor suppressor p16INK4a.

BACKGROUND: The four members of the INK4 gene family (p16(INK4a), p15(INK4b), p18(INK4c) and p19(INK4d)) inhibit the closely related cyclin-dependent kinases CDK4 and CDK6 as part of the regulation of the G1-->S transition in the cell-division cycle. Loss of INK4 gene product function, particularly that of p16(INK4a), is found in 10-60% of human tumors, suggesting that broadly applicable anticancer therapies might be based on restoration of p16(INK4a) CDK inhibitory function. Although much less frequent, defects of p19(INK4d) have also been associated with human cancer (osteosarcomas). The protein structures of some INK4 family members, determined by nuclear magnetic resonance (NMR) spectroscopy and X-ray techniques, have begun to clarify the functional role of p16(INK4a) and the dysfunction introduced by the mutations associated with human tumors. RESULTS: The crystal structure of human p19(INK4d) has been determined at 1.8 A resolution using multiple isomorphous replacement methods. The fold of p19(INK4d) produces an oblong molecule comprising five approximately 32-residue ankyrin-like repeats. The architecture of the protein demonstrates the high structural similarity within the INK4 family. Comparisons to other ankyrin-repeat-containing proteins (GABPbeta, 53BP2 and myotrophin) show similar structures with comparable hydrogen-bonding patterns and hydrophobic interactions. Such comparisons highlight the splayed beta-loop geometry that is specific to INK4 inhibitors. This geometry is the result of a modified ankyrin structure in the second repeat. CONCLUSIONS: Among the INK4 inhibitors, the highest amino acid sequence conservation is found in the helical stacks; this conservation creates a conserved beta-loop geometry specific to INK4 inhibitors. Therefore, in addition to models which predict that the conserved helix alpha6 is responsible for CDK inhibition, a binding mode whereby the loops of INK4 proteins bind to the CDKs should also be considered. A similar loop-based interaction is seen in the complex formed between the ankyrin-repeat-containing protein GABPbeta and_GABPalpha. This mode of binding would be consistent with the observation that p16(INK4a) is sensitive to deleterious mutations found throughout this tumor suppressor protein; these mutations probably destabilize the three-dimensional structure.

Amino Acid Sequence↗
PubMed1998

Structure of reduced DsbA from Escherichia coli in solution.

The three-dimensional structure of reduced DsbA from Escherichia coli in aqueous solution has been determined by nuclear magnetic resonance (NMR) spectroscopy and is compared with the crystal structure of oxidized DsbA [Guddat, L. W., Bardwell, J. C. A., Zander, T., and Martin, J. L. (1997) Protein Sci. 6, 1148-1156]. DsbA is a monomeric 21 kDa protein which consists of 189 residues and is required for disulfide bond formation in the periplasm of E. coli. On the basis of sequence-specific 1H NMR assignments, 1664 nuclear Overhauser enhancement distance constraints, 118 hydrogen bond distance constraints, and 293 dihedral angle constraints were obtained as the input for the structure calculations by simulated annealing with the program X-PLOR. The enzyme is made up of two domains. The catalytic domain has a thioredoxin-like fold with a five-stranded beta-sheet and three alpha-helices, and the second domain consists of four alpha-helices and is inserted into the thioredoxin motif. The active site between Cys30 and Cys33 is located at the N terminus of the first alpha-helix in the thioredoxin-like domain. The solution structure of reduced DsbA is rather similar to the crystal structure of the oxidized enzyme but exhibits a different relative orientation of both domains. In addition, the conformations of the active site and a loop between strand beta5 and helix alpha7 are slightly different. These structural differences may reflect important functional requirements in the reaction cycle of DsbA as they appear to facilitate the release of oxidized polypeptides from reduced DsbA. The extremely low pKa value of the nucleophilic active site thiol of Cys30 in reduced DsbA is most likely caused by its interactions with the dipole of the active site helix and the side chain of His32, as no other charged residues are located next to the sulfur atom of Cys30 in the solution structure.

Amino Acid Sequence↗
PubMed1998

Backbone dynamics of the CDK inhibitor p19(INK4d) studied by 15N NMR relaxation experiments at two field strengths.

The four members of the INK4 gene family, p16(INK4a), p15(INK4b), p18(INK4c) and p19(INK4d), are known to bind to and inhibit the closely related cyclin-dependent kinases CDK4 and CDK6 as part of the regulation of the G1/S transition in the cell division cycle. Loss of INK4 gene product function, and particularly that of p16(INK4a), is found in human cancer. 15N NMR relaxation rates of p19(INK4d) were analyzed using the reduced spectral density mapping method. Most of the backbone of p19(INK4d) exists in a well-defined structure of limited conformational flexibility on the nanosecond to picosecond time-scales. Introducing appropriate scaling to account for the effects of anisotropy, a considerable amount of exchange broadening was found for several residues throughout the sequence, especially residues in the second ankyrin repeat and in the beginnings and ends of loops connecting ankyrin repeats. A possible mode of binding between p19(INK4d) and CDK4 and CDK6 could therefore involve the loop segments of p19(INK4d). The average overall correlation time taumeff was determined to be 13.6 ns, reflecting the tendency of p19(INK4d) to aggregate.

Anisotropy↗
PubMed1998

An adiabatic multiple spin-echo pulse sequence: removal of systematic errors due to pulse imperfections and off-resonance effects.

Application of AFP (adiabatic fast passage) pulses for removal of systematic errors associated with multiple spin-echo sequences is demonstrated. The adiabatic fast passage pulses facilitate minimization of cumulative pulse errors for all three components of magnetization. It is also shown that off-resonance effects present in conventional CPMG sequences which degrade image quality in magnetic resonance imaging and introduce systematic errors in measured T2 relaxation time peak amplitudes can be suppressed by introduction of AFP pulses without any degradation of overall signal intensity. The technique has been tested on the 15N spin-spin relaxation time measurements of a 110 amino acid domain of the F-actin cross-linking protein.

Actins↗
PubMed1998

Parallel pathways in the folding of a short-term denatured scFv fragment of an antibody.

BACKGROUND: Antibodies are prototypes of multimeric proteins and consist of structurally similar domains. The two variable domains of an antibody (VH and VL) interact through a large hydrophobic interface and can be expressed as covalently linked single-chain Fv (scFv) fragments. The in vitro folding of scFv fragments after long-term denaturation in guanidinium chloride is known to be slow. In order to delineate the nature of the rate-limiting step, the folding of the scFv fragment of an antibody after short-term denaturation has been investigated. RESULTS: Secondary structure formation, measured by H/D-exchange protection, of a mutant scFv fragment of an antibody after short incubation in 6 M guanidinium chloride was shown to be multiphasic. NMR analysis shows that an intermediate with significant proton protection is observed within the dead time of the manual mixing experiments. Subsequently, the folding reaction proceeds via a biphasic reaction and mass spectrometry analyses of the exchange experiments confirm the existence of two parallel pathways. In the presence of cyclophilin, however, the faster of the two phases vanishes (when followed by intrinsic tryptophan fluorescence), while the slower phase is not significantly enhanced by equimolar cyclophilin. CONCLUSIONS: The formation of an early intermediate, which shows amide-proton exchange protection, is independent of proline isomerization. Subsequently, a proline cis-trans isomerization reaction in the rapidly formed intermediate, producing 'non-native' isomers, competes with the fast formation of native species. Interface formation in a folding intermediate of the scFv fragment is proposed to prevent the back-isomerization of these prolines from being efficiently catalyzed by cyclophilin.

Immunoglobulin Fragments↗