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Papers by justyna stec-niemczyk

Research paper thumbnail of Functional and Structural Characterization of Spl Proteases from Staphylococcus aureus

Journal of Molecular Biology, 2006

We have identified YkbA from Bacillus subtilis as a novel member of the L-amino acid transporter ... more We have identified YkbA from Bacillus subtilis as a novel member of the L-amino acid transporter (LAT) family of amino acid transporters. The protein is ϳ30% identical in amino acid sequence to the light subunits of human heteromeric amino acid transporters. Purified His-tagged YkbA from Escherichia coli membranes reconstituted in proteoliposomes exhibited sodium-independent, obligatory exchange activity for L-serine and L-threonine and also for aromatic amino acids, albeit with less activity. Thus, we propose that YkbA be renamed SteT (Ser/Thr exchanger transporter). Kinetic analysis supports a sequential mechanism of exchange for SteT. Freeze-fracture analysis of purified, functionally active SteT in proteoliposomes, together with blue native polyacrylamide gel electrophoresis and transmission electron microscopy of detergent-solubilized purified SteT, suggest that the transporter exists in a monomeric form.

Research paper thumbnail of A Novel Class of Cysteine Protease Inhibitors: Solution Structure of Staphostatin A from Staphylococcus aureus

Biochemistry, 2003

A series of secreted proteases are included among the virulence factors documented for Staphyloco... more A series of secreted proteases are included among the virulence factors documented for Staphylococcus aureus. In light of increasing antibiotic resistance of this dangerous human pathogen, these proteases are considered as suitable targets for the development of novel therapeutic strategies. The recent discovery of staphostatins, endogenous, highly specific, staphylococcal cysteine protease inhibitors, opened a possibility for structure-based design of low molecular weight analogues. Moreover, the crystal structure of staphostatin B revealed a distinct folding pattern and an unexpected, substrate-like binding mode. The solution structure of staphostatin A reported here confirms that staphostatins constitute a novel, distinct class of cysteine protease inhibitors. In addition, the structure knowledge-based mutagenesis studies shed light on individual structural features of staphostatin A, the inhibition mechanism, and the determinants of distinct specificity of staphostatins toward their target proteases.

Research paper thumbnail of Characterisation of a highly specific, endogenous inhibitor of cysteine protease from Staphylococcus epidermidis, a new member of the staphostatin family

Biological Chemistry, 2004

Staphostatins, a novel family of cysteine protease inhibitors with a unique mechanism of action a... more Staphostatins, a novel family of cysteine protease inhibitors with a unique mechanism of action and distinct protein fold has recently been discovered. In this report we describe the properties of Staphylococcus epidermidis staphostatin A (EcpB), a new member of the family. As for other staphostatins, the recombinant S. epidermidis staphostatin A exerted very narrow inhibitory specificity, limited to cysteine protease from the same species. The closely related proteases from S. aureus cleaved the inhibitor at the reactive site peptide bond and inactivated it. The EcpB homologue, S. aureus staphostatin A (ScpB), was also susceptible to proteolytic cleavage at the same site by non-target cysteine proteases. Conversely, S. aureus staphostatin B (SspC) was resistant to such proteolysis. The difference in the susceptibility of individual inhibitors to proteolytic cleavage at the reactive site suggests subtle variations in the mechanism of interaction with cysteine proteases.

Research paper thumbnail of Structural and functional characterization of SplA, an exclusively specific protease of Staphylococcus aureus

Biochemical Journal, 2009

Staphylococcus aureus is a dangerous human pathogen whose antibiotic resistance is steadily incre... more Staphylococcus aureus is a dangerous human pathogen whose antibiotic resistance is steadily increasing and no efficient vaccine is as yet available. This serious threat drives extensive studies on staphylococcal physiology and pathogenicity pathways, especially virulence factors. Spl (serine protease-like) proteins encoded by an operon containing up to six genes are a good example of poorly characterized secreted proteins probably involved in virulence. In the present study, we describe an efficient heterologous expression system for SplA and detailed biochemical and structural characterization of the recombinant SplA protease. The enzyme shares a significant sequence homology to V8 protease and epidermolytic toxins which are well documented staphylococcal virulence factors. SplA has a very narrow substrate specificity apparently imposed by the precise recognition of three amino acid residues positioned N-terminal to the hydrolysed peptide bond. To explain determinants of this extended specificity we resolve the crystal structure of SplA and define the consensus model of substrate binding. Furthermore we demonstrate that artificial N-terminal elongation of mature SplA mimicking a naturally present signal peptide abolishes enzymatic activity. The probable physiological role of the process is discussed. Of interest, even though precise N-terminal trimming is a common regulatory mechanism among S1 family enzymes, the crystal structure of SplA reveals novel significantly different mechanistic details.

Research paper thumbnail of Enzymatic Activity of the Staphylococcus aureus SplB Serine Protease is Induced by Substrates Containing the Sequence Trp-Glu-Leu-Gln

Journal of Molecular Biology, 2008

Proteases are of significant importance for the virulence of Staphylococcus aureus. Nevertheless,... more Proteases are of significant importance for the virulence of Staphylococcus aureus. Nevertheless, their subset, the serine protease-like proteins, remains poorly characterized. Here presented is an investigation of SplB protease catalytic activity revealing that the enzyme possesses exquisite specificity and only cleaves efficiently after the sequence Trp-Glu-Leu-Gln. To understand the molecular basis for such selectivity, we solved the threedimensional structure of SplB to 1.8 Å. Modeling of substrate binding to the protease demonstrated that selectivity relies in part on a canonical specificity pockets-based mechanism. Significantly, the conformation of residues that ordinarily form the oxyanion hole, an essential structural element of the catalytic machinery of serine proteases, is not canonical in the SplB structure. We postulate that within SplB, the oxyanion hole is only formed upon docking of a substrate containing the consensus sequence motif. It is suggested that this unusual activation mechanism is used in parallel with classical determinants to further limit enzyme specificity. Finally, to guide future development, we attempt to point at likely physiological substrates and thus the role of SplB in staphylococcal physiology.

Research paper thumbnail of The staphostatin family of cysteine protease inhibitors in the genus Staphylococcus as an example of parallel evolution of protease and inhibitor specificity

Biological Chemistry, 2007

Staphostatins constitute a family of staphylococcal cysteine protease inhibitors sharing a lipoca... more Staphostatins constitute a family of staphylococcal cysteine protease inhibitors sharing a lipocalin-like fold and a unique mechanism of action. Each of these cytoplasmic proteins is co-expressed from one operon, together with a corresponding target extracellular cysteine protease (staphopain). To cast more light on staphostatin/staphopain interaction and the evolution of the encoding operons, we have cloned and characterized a staphopain (StpA2 aur CH-91 ) and its inhibitor (StpinA2 aur CH-91 ) from a novel staphylococcal thiol protease operon (stpAB2 CH-91 ) identified in S. aureus strain CH-91. Furthermore, we have expressed a staphostatin from Staphylococcus warneri (StpinB war ) and characterized its target protease (StpB war ). Analysis of the reciprocal interactions among novel and previously described members of the staphostatin and staphopain families demonstrates that the cotranscribed protease is the primary target for each staphostatin. Nevertheless, the inhibitor derived from one species of Staphylococcus can inhibit the staphopain from another species, although the K i values are generally higher and inhibition only occurs if both proteins belong to the same subgroup of either S. aureus staphopain A/staphostatin A (a group) or staphopain B/staphostatin B (b group) orthologs. This indicates that both subgroups arose in a single event of ancestral allelic duplication, followed by parallel evolution of the protease/inhibitor pairs. The tight coevolution is likely the result of the known deleterious effects of uncontrolled staphopain action.

Research paper thumbnail of Modes of inhibition of cysteine proteases

Cysteine proteases are involved in many physiological processes and their hyperactivity may lead ... more Cysteine proteases are involved in many physiological processes and their hyperactivity may lead to severe diseases. Nature has developed various strategies to protect cells and whole organisms against undesired proteolysis. One of them is the control of proteolytic activity by inhibition. This paper presents the mechanisms underlying the action of proteinaceous inhibitors of cysteine proteinases and covers propeptides binding backwards relative to the substrate or distorting the protease catalytic centre similarly to serpins, the p35 protein binding covalently to the enzyme, and cystatins that are exosite binding inhibitors. The paper also discusses tyropins and chagasins that, although unrelated to cystatins, inhibit cysteine proteinases by a similar mechanism, as well as inhibitors of the apoptosis protein family that bind in a direction opposite to that of the substrate, similarly to profragments. Special attention is given to staphostatins, a novel family of inhibitors acting in an unusual manner.

Research paper thumbnail of Functional and Structural Characterization of Spl Proteases from Staphylococcus aureus

Journal of Molecular Biology, 2006

We have identified YkbA from Bacillus subtilis as a novel member of the L-amino acid transporter ... more We have identified YkbA from Bacillus subtilis as a novel member of the L-amino acid transporter (LAT) family of amino acid transporters. The protein is ϳ30% identical in amino acid sequence to the light subunits of human heteromeric amino acid transporters. Purified His-tagged YkbA from Escherichia coli membranes reconstituted in proteoliposomes exhibited sodium-independent, obligatory exchange activity for L-serine and L-threonine and also for aromatic amino acids, albeit with less activity. Thus, we propose that YkbA be renamed SteT (Ser/Thr exchanger transporter). Kinetic analysis supports a sequential mechanism of exchange for SteT. Freeze-fracture analysis of purified, functionally active SteT in proteoliposomes, together with blue native polyacrylamide gel electrophoresis and transmission electron microscopy of detergent-solubilized purified SteT, suggest that the transporter exists in a monomeric form.

Research paper thumbnail of A Novel Class of Cysteine Protease Inhibitors: Solution Structure of Staphostatin A from Staphylococcus aureus

Biochemistry, 2003

A series of secreted proteases are included among the virulence factors documented for Staphyloco... more A series of secreted proteases are included among the virulence factors documented for Staphylococcus aureus. In light of increasing antibiotic resistance of this dangerous human pathogen, these proteases are considered as suitable targets for the development of novel therapeutic strategies. The recent discovery of staphostatins, endogenous, highly specific, staphylococcal cysteine protease inhibitors, opened a possibility for structure-based design of low molecular weight analogues. Moreover, the crystal structure of staphostatin B revealed a distinct folding pattern and an unexpected, substrate-like binding mode. The solution structure of staphostatin A reported here confirms that staphostatins constitute a novel, distinct class of cysteine protease inhibitors. In addition, the structure knowledge-based mutagenesis studies shed light on individual structural features of staphostatin A, the inhibition mechanism, and the determinants of distinct specificity of staphostatins toward their target proteases.

Research paper thumbnail of Characterisation of a highly specific, endogenous inhibitor of cysteine protease from Staphylococcus epidermidis, a new member of the staphostatin family

Biological Chemistry, 2004

Staphostatins, a novel family of cysteine protease inhibitors with a unique mechanism of action a... more Staphostatins, a novel family of cysteine protease inhibitors with a unique mechanism of action and distinct protein fold has recently been discovered. In this report we describe the properties of Staphylococcus epidermidis staphostatin A (EcpB), a new member of the family. As for other staphostatins, the recombinant S. epidermidis staphostatin A exerted very narrow inhibitory specificity, limited to cysteine protease from the same species. The closely related proteases from S. aureus cleaved the inhibitor at the reactive site peptide bond and inactivated it. The EcpB homologue, S. aureus staphostatin A (ScpB), was also susceptible to proteolytic cleavage at the same site by non-target cysteine proteases. Conversely, S. aureus staphostatin B (SspC) was resistant to such proteolysis. The difference in the susceptibility of individual inhibitors to proteolytic cleavage at the reactive site suggests subtle variations in the mechanism of interaction with cysteine proteases.

Research paper thumbnail of Structural and functional characterization of SplA, an exclusively specific protease of Staphylococcus aureus

Biochemical Journal, 2009

Staphylococcus aureus is a dangerous human pathogen whose antibiotic resistance is steadily incre... more Staphylococcus aureus is a dangerous human pathogen whose antibiotic resistance is steadily increasing and no efficient vaccine is as yet available. This serious threat drives extensive studies on staphylococcal physiology and pathogenicity pathways, especially virulence factors. Spl (serine protease-like) proteins encoded by an operon containing up to six genes are a good example of poorly characterized secreted proteins probably involved in virulence. In the present study, we describe an efficient heterologous expression system for SplA and detailed biochemical and structural characterization of the recombinant SplA protease. The enzyme shares a significant sequence homology to V8 protease and epidermolytic toxins which are well documented staphylococcal virulence factors. SplA has a very narrow substrate specificity apparently imposed by the precise recognition of three amino acid residues positioned N-terminal to the hydrolysed peptide bond. To explain determinants of this extended specificity we resolve the crystal structure of SplA and define the consensus model of substrate binding. Furthermore we demonstrate that artificial N-terminal elongation of mature SplA mimicking a naturally present signal peptide abolishes enzymatic activity. The probable physiological role of the process is discussed. Of interest, even though precise N-terminal trimming is a common regulatory mechanism among S1 family enzymes, the crystal structure of SplA reveals novel significantly different mechanistic details.

Research paper thumbnail of Enzymatic Activity of the Staphylococcus aureus SplB Serine Protease is Induced by Substrates Containing the Sequence Trp-Glu-Leu-Gln

Journal of Molecular Biology, 2008

Proteases are of significant importance for the virulence of Staphylococcus aureus. Nevertheless,... more Proteases are of significant importance for the virulence of Staphylococcus aureus. Nevertheless, their subset, the serine protease-like proteins, remains poorly characterized. Here presented is an investigation of SplB protease catalytic activity revealing that the enzyme possesses exquisite specificity and only cleaves efficiently after the sequence Trp-Glu-Leu-Gln. To understand the molecular basis for such selectivity, we solved the threedimensional structure of SplB to 1.8 Å. Modeling of substrate binding to the protease demonstrated that selectivity relies in part on a canonical specificity pockets-based mechanism. Significantly, the conformation of residues that ordinarily form the oxyanion hole, an essential structural element of the catalytic machinery of serine proteases, is not canonical in the SplB structure. We postulate that within SplB, the oxyanion hole is only formed upon docking of a substrate containing the consensus sequence motif. It is suggested that this unusual activation mechanism is used in parallel with classical determinants to further limit enzyme specificity. Finally, to guide future development, we attempt to point at likely physiological substrates and thus the role of SplB in staphylococcal physiology.

Research paper thumbnail of The staphostatin family of cysteine protease inhibitors in the genus Staphylococcus as an example of parallel evolution of protease and inhibitor specificity

Biological Chemistry, 2007

Staphostatins constitute a family of staphylococcal cysteine protease inhibitors sharing a lipoca... more Staphostatins constitute a family of staphylococcal cysteine protease inhibitors sharing a lipocalin-like fold and a unique mechanism of action. Each of these cytoplasmic proteins is co-expressed from one operon, together with a corresponding target extracellular cysteine protease (staphopain). To cast more light on staphostatin/staphopain interaction and the evolution of the encoding operons, we have cloned and characterized a staphopain (StpA2 aur CH-91 ) and its inhibitor (StpinA2 aur CH-91 ) from a novel staphylococcal thiol protease operon (stpAB2 CH-91 ) identified in S. aureus strain CH-91. Furthermore, we have expressed a staphostatin from Staphylococcus warneri (StpinB war ) and characterized its target protease (StpB war ). Analysis of the reciprocal interactions among novel and previously described members of the staphostatin and staphopain families demonstrates that the cotranscribed protease is the primary target for each staphostatin. Nevertheless, the inhibitor derived from one species of Staphylococcus can inhibit the staphopain from another species, although the K i values are generally higher and inhibition only occurs if both proteins belong to the same subgroup of either S. aureus staphopain A/staphostatin A (a group) or staphopain B/staphostatin B (b group) orthologs. This indicates that both subgroups arose in a single event of ancestral allelic duplication, followed by parallel evolution of the protease/inhibitor pairs. The tight coevolution is likely the result of the known deleterious effects of uncontrolled staphopain action.

Research paper thumbnail of Modes of inhibition of cysteine proteases

Cysteine proteases are involved in many physiological processes and their hyperactivity may lead ... more Cysteine proteases are involved in many physiological processes and their hyperactivity may lead to severe diseases. Nature has developed various strategies to protect cells and whole organisms against undesired proteolysis. One of them is the control of proteolytic activity by inhibition. This paper presents the mechanisms underlying the action of proteinaceous inhibitors of cysteine proteinases and covers propeptides binding backwards relative to the substrate or distorting the protease catalytic centre similarly to serpins, the p35 protein binding covalently to the enzyme, and cystatins that are exosite binding inhibitors. The paper also discusses tyropins and chagasins that, although unrelated to cystatins, inhibit cysteine proteinases by a similar mechanism, as well as inhibitors of the apoptosis protein family that bind in a direction opposite to that of the substrate, similarly to profragments. Special attention is given to staphostatins, a novel family of inhibitors acting in an unusual manner.