Effects of mutations in the hinge region of serpins

Hopkins, Paul C.R.; Carrell, Robin W.; Stone, Stuart R.

doi:10.1021/bi00081a008

Cited by 190 publications

(193 citation statements)

References 53 publications

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“…Among these regions the hinge is thought to provide the essential mobility required for conformational changes in the RCL during its incorporation into the serpin ␤-sheet A structural elements after proteolysis (8,22). The RCL is hyper-variable in its composition and length, and in the case of inhibitory serpins this is presumed to reflect an evolutionary adaptation for optimal interactions with their target proteinases (8,23).…”

Section: Discussionmentioning

confidence: 99%

Residues in the Human Corticosteroid-binding Globulin Reactive Center Loop That Influence Steroid Binding before and after Elastase Cleavage

Lin

Underhill

Gardill

et al. 2009

Journal of Biological Chemistry

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Corticosteroid-binding globulin (CBG) is a non-inhibitory serine proteinase inhibitor (serpin) that transports cortisol and progesterone in blood. Crystal structures of rat CBG and a thrombin-cleaved human CBG:anti-trypsin (Pittsburgh) chimera show how structural transitions after proteolytic cleavage of the CBG reactive center loop (RCL) could disrupt steroid binding. This ligand release mechanism is assumed to involve insertion of the cleaved RCL into the ␤-sheet A of the serpin structure. We have, therefore, examined how amino acid substitutions in the human CBG RCL influence steroid binding before and after its cleavage by neutrophil elastase. Elastase-cleaved wild-type CBG or variants with substitutions at P15 and/or P16 (E334G/G335N or E334A) lost steroid binding completely, whereas deletion of Glu-334 resulted in no loss of steroid binding after RCL cleavage, presumably because this prevents its insertion into ␤-sheet A. Similarly, the steroid binding properties of CBG variants with substitutions at P15 (G335P), P14 (V336R), or P12 (T338P) in the RCL hinge were largely unaffected after elastase cleavage, most likely because the re-orientation and/or insertion of the cleaved RCL was blocked. Substitutions at P10 (G340P, G340S) or P8 (T342P, T342N) resulted in a partial loss of steroid binding after proteolysis which we attribute to incomplete insertion of the cleaved RCL. Remarkably, several substitutions (E334A, V336R, G340S, and T342P) increased the steroid binding affinities of human CBG even before elastase cleavage, consistent with the concept that CBG normally toggles between a high affinity ligand binding state where the RCL is fully exposed and a lower affinity state in which the RCL is partly inserted into ␤-sheet A. Corticosteroid-binding globulin (CBG)2 is the major carrier protein for natural glucocorticoids (cortisol and corticosterone) and progesterone in blood (1), and it regulates the bioavailability of steroids that control numerous physiological processes including reproduction, inflammation, stress responses, and tissue development (2). Because CBG binds up to 90% of the glucocorticoids in blood plasma, it serves as a reservoir of anti-inflammatory steroids that can be released at their sites of action (3). The latter concept was proposed when it was discovered that human CBG exhibits remarkable sequence identity with the archetypal serine proteinase inhibitor, ␣1-anti-trypsin (AAT), and was based on the realization that CBG might also be a target of specific classes of proteinases (4).The close structural relationship between CBG and AAT defines it as a clade A serine proteinase inhibitor (serpin) family member (5). Many of these serpin A family members, including CBG, are encoded by genes in the human 14q21.1 chromosome cluster and are thought to have arisen by gene duplication to produce serpins with similar properties and physiological functions (6). Unlike most clade A serpins, CBG is not known to act as a proteinase inhibitor but appears to be a suicide substrate of specific protei...

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Section: Discussionmentioning

confidence: 99%

Residues in the Human Corticosteroid-binding Globulin Reactive Center Loop That Influence Steroid Binding before and after Elastase Cleavage

Lin

Underhill

Gardill

et al. 2009

Journal of Biological Chemistry

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“…This idea originally stems from the observation that the non-inhibitory ovalbumin has an arginine residue in the P,, position (Wright et al, 1990), the insertion of the charged and bulky arginine residue into the interior of the protein believed to be energetically unfavorable. Furthermore, substrate behaviour is exhibited by variants containing substitions with charged amino acids or proline in the hinge region (Caso et al, 1991;Skriver et al, 1991;Hopkins et al, 1993;Audenaert et al, 1994;Hood et al, 1994;Lawrence et al, 1994), and by binary complexes between a serpin and peptides corresponding to P,-P,, Schulze et al, 1990;Bjiirk et al, 1992). Two possible mechanisms for explaining the need for insertion have been discussed (Potempa et al, 1994).…”

Section: Discussionmentioning

confidence: 99%

Conformational Changes of the Reactive‐Centre Loop and β‐Strand 5A Accompany Temperature‐Dependent Inhibitor‐Substrate Transition of Plasminogen‐Activator Inhibitor 1

Kjøller

Martensen

Sottrup‐Jensen

et al. 1996

European Journal of Biochemistry

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We have studied conformational changes of type-I plasminogen-activator inhibitor (PAI-1) during a temperature-dependent inhibitor -substrate transition by measuring susceptibility of the molecule to nontarget proteinases. When incubated at 0°C instead of the normally used 37"C, a tenfold decrease in the specific inhibitory activity of active PAI-1 was observed. Accordingly, PAI-I was recovered in a reactivecentre-cleaved form from incubations with urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA) at 0"C, but not at 37°C. It thus behaved as a substrate for the target proteinases at the lower temperature. Active PAI-1 was exposed to a variety of non-target proteinases, including elastase, papain, thermolysin, trypsin, and V8 proteinase. It was found that specific peptide bonds in the reactive centre loop (RCL) and strand 5 in @-sheet A (sSA) had a temperature-dependent proteolytic susceptibility, while the P,,-P,, (E332-S333) bond, forming the hinge between s5A and the RCL, showed indistinguishable susceptibility to proteolysis by V8 proteinase at 0" and 37°C. In latent and reactivecentre-cleaved PAI-1, all the bonds were resistant to proteolysis at the higher as well as the lower temperature. An anti-PAI-I monoclonal antibody maintained the inhibitory activity of PAIL1 and prevented reactive centre cleavage at 0 "C, and thus prevented substrate behaviour. Concomitantly, it caused specific changes in proteolytic susceptibility of sSA and the RCL, but it did not affect cleavage of the P,,-PI, bond by V8 proteinase. Our observations suggest that temperature-dependent conformational changes of 8-sheet A and the RCL determine whether the serpin act as an inhibitor or a substrate. Furthermore they suggest that the RCL of PAI-I is fully extracted from 8-sheet A in the inhibitory as well as in the substrate form, favoring a so-called induced conformational state model to explain why inhibitory activity requires partial insertion of the RCL into 8-sheet A.Keywords: conformation ; plasminogen-activator inhibitor; proteolysis ; reactive-centre loop; serpin.The serpin superfamily includes of a large number serine proteinase inhibitors and a variety of proteins with other or unknown functions (see reviews by Huber and Carrell, 1989;Potempa et al., 1994). The molecular mechanism of the inhibitory function of all the serine proteinase inhibitor members of the superfamily is thought to be similar, on the basis of a high percentage of sequence identity and the same over-all fold, as shown by X-ray crystal analysis. A key feature is an approximately 20-amino-acid peptide segment, the reactive-centre loop (RCL) which protrudes from the main body of the molecule (see reviews by Bode and Huber, 1992; Stein and Carrell, 1995).

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“…Like other serpins, MENT possesses a reactive center loop (RCL) 2 through which interaction with a cognate proteinase occurs. The presence of an inhibitory hinge motif (12) in its RCL suggests that MENT may retain the propensity for serpin-like conformational transitions and hence proteinase inhibition.…”

Section: Ment (Myeloid and Erythroid Nuclear Termination Stage-specifmentioning

confidence: 99%

“…b, SDS-PAGE and Western blotting of the protein from the CV-1 nuclei (protein concentration 150 g/ml) without (Control) and with 50 g/ml E-64d (ϩ E-64d). c, SDS-PAGE and Western blotting of the protein from the nuclear extracts isolated from CV-1 cells transfected with cDNA encoding MENT WT (lanes 1-5), MENT P14R (lanes 6 -10), MENT OV (lanes [11][12][13][14][15], and control vector (lanes 16 -20) and incubated at 37°C for 0 min (lanes 1, 6, 11, and 16), 20 min (lanes 2, 7, 12, and 17), 1 h (lanes 3, 8, 13, and 18), 3 h (lanes 4, 9, 14, and 19) and 20 h (lanes 5, 10, 15, and 20). Western blots (b and c) were detected using antibodies against Rb.…”

Section: Figmentioning

confidence: 99%

Inhibitory Activity of a Heterochromatin-associated Serpin (MENT) against Papain-like Cysteine Proteinases Affects Chromatin Structure and Blocks Cell Proliferation

Irving

Шушанов

Pike

et al. 2002

Journal of Biological Chemistry

103

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MENT (Myeloid and Erythroid NuclearTermination stage-specific protein) is a developmentally regulated chromosomal serpin that condenses chromatin in terminally differentiated avian blood cells. We show that MENT is an effective inhibitor of the papain-like cysteine proteinases cathepsins L and V. In addition, ectopic expression of MENT in mammalian cells is apparently sufficient to inhibit a nuclear papain-like cysteine proteinase and prevent degradation of the retinoblastoma protein, a major regulator of cell proliferation. MENT also accumulates in the nucleus, causes a strong block in proliferation, and promotes condensation of chromatin. Variants of MENT with mutations or deletions within the M-loop, which contains a nuclear localization signal and an AT-hook motif, reveal that this region mediates nuclear transport and morphological changes associated with chromatin condensation. Noninhibitory mutants of MENT were constructed to determine whether its inhibitory activity has a role in blocking proliferation. These mutations changed the mode of association with chromatin and relieved the block in proliferation, without preventing transport to the nucleus. We conclude that the repressive effect of MENT on chromatin is mediated by its direct interaction with a nuclear protein that has a papain-like cysteine proteinase active site.MENT (Myeloid and Erythroid Nuclear Termination stagespecific protein), a developmentally regulated nuclear protein, is present in three main avian blood cell types (erythrocytes, lymphocytes, and granulocytes) where it is the predominant non-histone protein associated with compact heterochromatin (1). In vitro, MENT brings about a dramatic remodeling and condensation of chromatin higher order structure by forming protein "bridges" connecting separate nucleosomes in nucleosome arrays (for review see Ref.2). MENT has no homology with other known chromatin proteins but belongs to the intracellular branch of the serpin superfamily (3). Serpins were originally characterized as serine proteinase inhibitors; however, more recently certain members have been shown to be capable of inhibiting other proteinase classes such as caspases (the viral serpin crmA (4)) and papain-like cysteine proteinases (SCCA-1 (5)). The inhibitory members of the serpin family are notable for their ability to undergo a large scale conformational transition (for review see Ref. 6) that is critical for inhibition of target proteinases (7, 8) and for self-association or polymerization (9 -11). Multiple sequence alignments and comparison with known serpin structures reveal that MENT contains a large insertion, the "M-loop," between the C-and D-helices. This loop contains two critical functional motifs as follows: a classical nuclear localization signal (NLS) 1 that is required for nuclear import, and an AT-hook motif that is involved in chromatin and DNA binding (3). Like other serpins, MENT possesses a reactive center loop (RCL) 2 through which interaction with a cognate proteinase occurs. The presence of an inhibitory...

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Effects of mutations in the hinge region of serpins

Cited by 190 publications

References 53 publications

Residues in the Human Corticosteroid-binding Globulin Reactive Center Loop That Influence Steroid Binding before and after Elastase Cleavage

Residues in the Human Corticosteroid-binding Globulin Reactive Center Loop That Influence Steroid Binding before and after Elastase Cleavage

Conformational Changes of the Reactive‐Centre Loop and β‐Strand 5A Accompany Temperature‐Dependent Inhibitor‐Substrate Transition of Plasminogen‐Activator Inhibitor 1

Inhibitory Activity of a Heterochromatin-associated Serpin (MENT) against Papain-like Cysteine Proteinases Affects Chromatin Structure and Blocks Cell Proliferation

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