Prothrombin domains: circular dichroic evidence for a lack of cooperativity

Bloom, James W.; Mann, Kenneth G.

doi:10.1021/bi00577a017

Cited by 22 publications

(9 citation statements)

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“…Thus the environment experienced by DAPA bound to thrombin appears representative of that available to substrate, The differences in the fluorescence properties of a-thrombin-DAPA and prethrombin 2-DAPA undoubtedly reflect the differences between the configuration of the thrombin active site and its analogue in the zymogen prethrombin 2. Such structural differences are most likely of limited extent, however, as previous studies from this laboratory using circular dichroism spectroscopy (Bloom & Mann, 1979) indicate that no gross conformational changes occur upon cleavage of prothrombin to its activation peptides, intermediates, or Prothrombin Meizo-thrombin Fragment T2 + Prethrombin-2 Thrombin Fragment 1 plus Fragment 2 (cleavage products of Fragments 1-2 by thrombin) Fragment 1 plus Meizo-thrombin des Fragment 1 (result of autocatalysis from the second cleavage site. This can be prevented by the inhibitor, DAPA) figure 9: Prothrombin and its cleavage products.…”

Section: Discussionmentioning

confidence: 90%

Progressive development of a thrombin inhibitor binding site

Hibbard¹,

Nesheim²,

Mann³

1982

Biochemistry

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The studies reported here were undertaken to determine whether the thrombin precursors prothrombin, prethrombin 1, prethrombin 2, and Meizo thrombin interact with the fluorescent, reversible thrombin inhibitor dansylarginine N,N-(3-ethyl-1,5-pentanediyl)amide (DAPA) [Nesheim, M. E., Prendergast, F. G., & Mann, K. G. (1979) Biochemistry 18, 996--1003]. The results indicate that prothrombin and prethrombin 1, in which the cleavage sites at Arg274-Thr275 and Arg323-Ile324 both remain intact, do not bind DAPA, while prethrombin 2 or Meizo thrombin, which results respectively from a single cleavage of prothrombin at Arg274-Thr275 or Arg323-Ile324, do bind the inhibitor. Since prethrombin 2 is a precursor of thrombin without measurable enzymatic activity, a thorough characterization of its interaction with DAPA was undertaken. The interaction of DAPA with bovine thrombin similarly was studied for comparative purposes. The binding of DAPA to either protein is accompanied by changes in the fluorescence properties of the dansyl moiety including increases in emission intensity, excited-state lifetime, polarization, and a slight blue shift in the wavelength of maximum emission intensity. Corrected excitation spectra indicate energy transfer to DAPA from one or more aromatic side chains of both proteins. Values of P0 for both complexes were extrapolated from Perrin plots of polarization vs. temperature and suggest that the dansyl moiety is held more rigidly in thrombin than in prethrombin 2. With excitation at either 280 or 335 nm the emission intensity of DAPA-prethrombin 2 is substantially less than that of the DAPA-thrombin complex. In contrast, the intensity of the Meizo thrombin-DAPA complex is greater than that of the DAPA-thrombin complex. From measurements of intensity changes the dissociation constants and stoichiometry of DAPA binding to thrombin and prethrombin 2 were measured. Prethrombin 2 binds to DAPA with a Kd = 5.9 x 10(-7) M (n = 1) while thrombin binds about 30 times more tightly with a Kd = 2.0 x 10(-8) M (n = 1). The active site directed irreversible thrombin inhibitors diisopropyl phosphorofluoridate and D-phenylalanylprolylarginyl chloromethyl ketone displace DAPA from thrombin but not from prethrombin 2. The results of these studies indicate the binding of a presumed substrate analogue (DAPA) to an inactive zymogen, prethrombin 2. In addition, the lack of DAPA binding to prothrombin and prethrombin 1, under conditions in which it binds to prethrombin 2, implicates events that accompany cleavage at Arg274-Arg275 in the "progressive" formation of an active site, even though further cleavage at Arg323-Ile324 is required for expression of enzymatic activity.

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Section: Discussionmentioning

confidence: 90%

Progressive development of a thrombin inhibitor binding site

Hibbard¹,

Nesheim²,

Mann³

1982

Biochemistry

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“…This ity to suggest a shift in prothrombin conformation on makes it difficult to detect changes in the whole molebinding to PS/POPC membranes. On the other hand, cule (Nelsestuen, 1976;Predergast and Mann, 1977; binding to DOPG/POPC vesicles did not alter prothrom- Bloom and Mann, 1979;Marsh et al, 1979; see also bin secondary structure significantly relative to that Table 2 and below).…”

Section: Analysis Of Prothrombin Amide 1' Fsd Spectra and Secondary Smentioning

confidence: 93%

Fourier transform infrared spectroscopic study of Ca2+ and membrane-induced secondary structural changes in bovine prothrombin and prothrombin fragment 1

Wu¹,

Lentz²

1991

Biophysical Journal

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Fourier transform infrared (FTIR) spectroscopy was used to monitor secondary structural changes associated with binding of bovine prothrombin and prothrombin fragment 1 to acidic lipid membranes. Prothrombin and prothrombin fragment 1 were examined under four different conditions: in the presence of (a) Na2EDTA, (b) 5 mM CaCl2, and in the presence of CaCl2 plus membranes containing 1-palmitoyl-2-oleoyl-3-sn-phosphatidylcholine (POPC) in combination with either (c) bovine brain phosphatidyl-serine (bovPS) or (d) 1,2-dioleoyl-phosphatidylglycerol (DOPG). The widely reported Ca(2+)-induced conformational change in bovine prothrombin fragment 1 was properly detected by our procedures, although Ca(2+)-induced changes in whole prothrombin spectra were too small to be reliably interpreted. Binding of prothrombin in the presence of Ca2+ to procoagulant POPC/bovPS small unilamellar vesicles produced an increase in ordered secondary structures (2% and 3% increases in alpha-helix and beta-sheet, respectively) and a decrease of random structure (5%) as revealed by spectral analysis on both the original and Fourier-self-deconvolved data and by difference spectroscopy with the undeconvolved spectra. Binding to POPC/DOPG membranes, which are less active as procoagulant membranes, produced no detectable changes in secondary structure. In addition, no change in prothrombin fragment 1 secondary structure was detectable upon binding to either POPC/bovPS or POPC/DOPG membranes. This indicates that a membrane-induced conformational change occurs in prothrombin in the nonmembrane-binding portion of the molecule, part of which is activated to form thrombin, rather than in the membrane-binding fragment 1 region. The possible significance of this conformational change is discussed in terms of differences between the procoagulant activities of different acidic lipid membranes.

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“…By limited proteolysis, a technique suitable to detect structural domains of proteins, it was shown that both prothrombin and plasminogen may be dissected at the boundaries separating kringles, while leaving the kringles intact (1,2). Circular dichroism studies on prothrombin and its proteolytic fragments showed that little alteration occurs in the structure of kringles after isolation of the fragments, suggesting that in prothrombin the kringles exist as distinct, noninteracting structural domains (14). Differential scanning calorimetry of plasminogen, prothrombin, and their proteolytic fragments revealed that the AH value of the thermal transition of intact prothrombin or plasminogen is the sum of the AH value of the thermal transitions of the individual fragments, indicating that the kringles exist as independent domains (15,16).…”

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confidence: 99%

Folding autonomy of the kringle 4 fragment of human plasminogen.

Trexler

Patthy

1983

Proc. Natl. Acad. Sci. U.S.A.

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Kringle 4, an 88-residue plasminogen fragment carrying a lysine-binding site, loses its affinity for lysine-Sepharose upon reductive cleavage of its disulfide bridges. Aerobic incubation of the reduced, denatured fragment results in the rapid restoration of the disulfide bonds with concomitant recovery of lysine-Sepharose affinity. The ability of the unfolded fragment to regain its native conformation suggests that the kringle structure is an autonomous folding domain. During refolding of kringle 4 the native disulfide bonds, Cys22 Cys62 and Cys50 Cys74, appear first. The folding intermediate possessing these two disulfide bridges already binds to lysine-Sepharose, indicating that the third native bridge, which in native kringle 4 connects residues Cys' and Cys79, is not essential for the maintenance of the biologically active conformation of kringle 4. Comparison of the sequences of human prothrombin, urokinase, and plasminogen kringles revealed that the residues surrounding the (Cys22 Cys62 and Cys5" Cys74 bridges constitute the most conservative segments of kringles, whereas the residues neighboring the Cys' Cys79 bridge are not highly conserved. We propose that conservation of various residues in the different kringles reflects their importance for the folding autonomy of kringles.Plasminogen, prothrombin, and urokinase, unlike the proenzymes of pancreatic serine proteases, possess large protein extensions at the amino-terminal end of the homologous protease part (1-4). These nonprotease segments are important for the biological specificity and control ofplasmin, thrombin, and urokinase because they are involved in interactions that regulate plasminogen activation and plasmin-catalyzed fibrinolysis (5) or control the prothrombin-thrombin conversion (6). Thus, the nonprotease regions participate in the Ca2+-mediated binding of prothrombin to phospholipid membranes, in the association of prothrombin with factor Va (7,8), and in the interactions of plasmin(ogen) with fibrin, a2-antiplasmin, and w-aminocarboxylic acids (9-11).The multiplicity of binding functions associated with the nonprotease segments of prothrombin, plasminogen, and urokinase is reflected in the structure of these proteins inasmuch as these regions are divided into discrete structural-functional units. In the case of plasminogen five triple-loop, three-disulfide-bridge "kringles" are discernible in the nonprotease segment (1). The five structural units display identical gross architecture and remarkable sequence homology, making the conclusion inescapable that these units arose as a result of 5-fold internal repetition of the same gene piece (12, 13). The aminoterminal part of prothrombin also contains two kringles, closely homologous to those of plasminogen (2, 3); the kringles of plasminogen and prothrombin are in fact more closely related than their protease parts (12). Urokinase was also shown recently to possess a kringle structure that shows extensive homology with the prothrombin and plasminogen kringles (4).

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Prothrombin domains: circular dichroic evidence for a lack of cooperativity

Cited by 22 publications

References 22 publications

Progressive development of a thrombin inhibitor binding site

Progressive development of a thrombin inhibitor binding site

Fourier transform infrared spectroscopic study of Ca2+ and membrane-induced secondary structural changes in bovine prothrombin and prothrombin fragment 1

Folding autonomy of the kringle 4 fragment of human plasminogen.

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