Crystal structure of the apoptosis-inducing human granzyme A dimer

Granzyme A (GzmA) belongs to a family of trypsin-like serine proteases localized in cytoplasmic granules of activated lymphocytes and natural killer (NK) cells. In contrast to the related granzyme B (GzmB), GzmA forms a stable disulfide-linked homodimer and triggers target-cell death in a caspase-independent way. Limited proteolysis of a high-molecular-mass complex containing SET (also named putative HLA-associated protein II or PHAPII), PHAPI (pp32, leucine-rich acidic nuclear protein) and HMG2 by GzmA liberates NM23-H1, a Mg2+-dependent DNase that causes single-stranded breaks in nuclear DNA. By analyzing the dimeric GzmA structure at a resolution of 2.5 Angstrom, we determined the substrate-binding constraints and selective advantages of the two domains arranged as a unique functional tandem. The active sites of the two subunits point in opposite directions and the nearby noncatalytic surfaces can function as exosites, presenting substrates to the active site region of the adjacent partner in a manner analogous to staphylokinase or streptokinase, which present plasminogen to the cofactor plasmin and cofactor plasminogen complexes

Crystal structure of the apoptosis-inducing human granzyme A dimer

Granzyme A (GzmA) belongs to a family of trypsin-like serine proteases localized in cytoplasmic granules of activated lymphocytes and natural killer (NK) cells. In contrast to the related granzyme B (GzmB), GzmA forms a stable disulfide-linked homodimer and triggers target-cell death in a caspase-independent way. Limited proteolysis of a high-molecular-mass complex containing SET (also named putative HLA-associated protein II or PHAPII), PHAPI (pp32, leucine-rich acidic nuclear protein) and HMG2 by GzmA liberates NM23-H1, a Mg2+-dependent DNase that causes single-stranded breaks in nuclear DNA. By analyzing the dimeric GzmA structure at a resolution of 2.5 Angstrom, we determined the substrate-binding constraints and selective advantages of the two domains arranged as a unique functional tandem. The active sites of the two subunits point in opposite directions and the nearby noncatalytic surfaces can function as exosites, presenting substrates to the active site region of the adjacent partner in a manner analogous to staphylokinase or streptokinase, which present plasminogen to the cofactor plasmin and cofactor plasminogen complexes

Crystal structure of the apoptosis-inducing human granzyme A dimer

Granzyme A (GzmA) belongs to a family of trypsin-like serine proteases localized in cytoplasmic granules of activated lymphocytes and natural killer (NK) cells. In contrast to the related granzyme B (GzmB), GzmA forms a stable disulfide-linked homodimer and triggers target-cell death in a caspase-independent way. Limited proteolysis of a high-molecular-mass complex containing SET (also named putative HLA-associated protein II or PHAPII), PHAPI (pp32, leucine-rich acidic nuclear protein) and HMG2 by GzmA liberates NM23-H1, a Mg2+-dependent DNase that causes single-stranded breaks in nuclear DNA. By analyzing the dimeric GzmA structure at a resolution of 2.5 Angstrom, we determined the substrate-binding constraints and selective advantages of the two domains arranged as a unique functional tandem. The active sites of the two subunits point in opposite directions and the nearby noncatalytic surfaces can function as exosites, presenting substrates to the active site region of the adjacent partner in a manner analogous to staphylokinase or streptokinase, which present plasminogen to the cofactor plasmin and cofactor plasminogen complexes

The 2.2-angstrom crystal structure of human pro-granzyme K reveals a rigid zymogen with unusual features

Granzyme K (GzmK) belongs to a family of trypsin-like serine proteases localized in electron dense cytoplasmic granules of activated natural killer and cytotoxic T-cells. Like the related granzymes A and B, GzmK can trigger DNA fragmentation and is involved in apoptosis. We expressed the Ser(195)-->Ala variant of human pro-GzmK in Escherichia coli, crystallized it, and determined its 2.2-Angstrom x-ray crystal structure. Pro-GzmK possesses a surprisingly rigid structure, which is most similar to activated serine proteases, in particular complement factor D, and not their proforms. The N-terminal peptide Met(14)-Ile(17) projects freely into solution and can be readily approached by cathepsin C, the natural convertase of pro-granzymes. The pre-shaped S1 pocket is occupied by the ion paired residues Lys(181B)-Asp(194) and is hence not available for proper substrate binding. The Ser(214)-Cys(220) segment, which normally provides a template for substrate binding, bulges out of the active site and is distorted. With analogy to complement factor D, we suggest that this strand will maintain its nonproductive conformation in mature GzmK, mainly due to the unusual residues Gly(215), Glu(219), and Val(94). We hypothesize that GzmK is proteolytically active only toward specific, as yet unidentified substrates, which upon approach transiently induce a functional active-site conformation. [References: 62

Differential expression of the granzymes A, K and M and perforin in human peripheral blood lymphocytes

Granzymes (Gzm) are a group of serine proteases which are stored in the granules of cytotoxic lymphocytes. In humans, five granzymes have been characterized to date at the molecular level. While GzmA and GzmB have been extensively studied, little is known about GzmH, GzmK and GzmM. In this study, we describe the generation of mAbs against human GzmK and GzmM by genetic immunization. The obtained anti-GzmK and anti-GzmM mAbs are not cross-reactive with GzmA, GzmB, GzmM and GzmA, GzmB, GzmK, respectively, and show a granular staining pattern in human lymphocytes. Flow cytometric analysis of peripheral blood lymphocytes revealed that GzmA, GzmM and perforin show a similar distribution. They are expressed in almost all CD161CD561 NK cells, CD31CD561 NKT cells and cd T cells as well as in 20–30 % of all CD31CD81 TC cells. Surprisingly, GzmK was not detected in the highly cytotoxic CD161CD561 NK cells but was preferentially expressed in lymphocytes of the T cell lineage, staining 20 % of CD31CD81 TC cells, 50 % of CD3 1CD561 NKT cells and 40 % of cd T cells, as well as 60 % of the small sub-population of CD56bright1 NK cells. Our data suggest that human granzymes are differentially expressed in distinct sub-populations of peripheral blood lymphocytes

The 2.2-angstrom crystal structure of human pro-granzyme K reveals a rigid zymogen with unusual features

Granzyme K (GzmK) belongs to a family of trypsin-like serine proteases localized in electron dense cytoplasmic granules of activated natural killer and cytotoxic T-cells. Like the related granzymes A and B, GzmK can trigger DNA fragmentation and is involved in apoptosis. We expressed the Ser(195)-->Ala variant of human pro-GzmK in Escherichia coli, crystallized it, and determined its 2.2-Angstrom x-ray crystal structure. Pro-GzmK possesses a surprisingly rigid structure, which is most similar to activated serine proteases, in particular complement factor D, and not their proforms. The N-terminal peptide Met(14)-Ile(17) projects freely into solution and can be readily approached by cathepsin C, the natural convertase of pro-granzymes. The pre-shaped S1 pocket is occupied by the ion paired residues Lys(181B)-Asp(194) and is hence not available for proper substrate binding. The Ser(214)-Cys(220) segment, which normally provides a template for substrate binding, bulges out of the active site and is distorted. With analogy to complement factor D, we suggest that this strand will maintain its nonproductive conformation in mature GzmK, mainly due to the unusual residues Gly(215), Glu(219), and Val(94). We hypothesize that GzmK is proteolytically active only toward specific, as yet unidentified substrates, which upon approach transiently induce a functional active-site conformation. [References: 62