Page 3 - U. Protein structure and function

Binding of Glutathione and ppGpp to Stringent Starvation Protein A (SspA)

Taner Duysak and Che-Hun Jung

Department of Molecular Medicine, Department of Chemistry

Graduate School, Chonnam National University, Gwangju, Korea

(Abstract)

Stringent starvation protein A (SspA) is a glutathione S-transferase homolog. In this

study, his6-tagged SspA from Escherichia coli has been cloned and over-expressed.

SspA binds glutathione and 1-chloro-2,4-dinitrobenzene, the substrates for glutathione

S-transferases, with the dissociation constants as 225.0 ± 34.4 μM and 75.3 ± 4.3 μM,

respectively. This observation is contradictory to the previous report that SspA, lacking

glutathione S-transferase activity, does not bind glutathione. It has been reported that

SspA is an RNA polymerase-associated transcription factor and that a functional relA

gene is required for SspA to affect gene expression. A function of relA is to synthesize

ppGpp, a global regulator in replication, transcription, and translation. This study

shows for the first time that SspA binds ppGpp with the dissociation of constants of

109.1 ± 7.2 μM. This study may provide an insight why relA is required for regulating

gene expression by SspA.

Figure 4. Determination of the dissociation constant (KD) for CDNB on SspA by

Sequence alignment of E. coli SspA and GstA. fluorescence spectroscopy. (a) The fluorescence spectra of SspA in the presence of

various concentrations of CDNB. While excited at 280 nm, the emission spectra of SspA

SspA MAVAANKRSVMTLFS--GPTDIYSHQVRIVLAEKGVSFEIEHVE----KDNPPQDL (10 μg/mL) were recorded at 25 ⁰C. (b) The fluorescence changes (ΔF) at 330 nm against

M LF G + SH I L E G F + V+ + D CDNB concentrations.

GstA ----------MKLFYKPGACSLASH---ITLRESGKDFTLVSVDLMKKRLENGDDY

Binding of ppGpp to SspA

SspA IDLNPNQSVPTLVDRELTLW-ESRIIMEYLDERFPHPPLM-PVYPVARGESRLYMH

+NP VP L+ + TL E IM+YL + P L+ PV ++R ++ +++

GstA FAVNPKGQVPALLLDDGTLLTEGVAIMQYLADSVPDRQLLAPVNSISRYKTIEWLN SspA seems not to be required for normal growth conditions for bacteria, and its

synthesis is dramatically stimulated by a stringent response. It is also reported that a

SspA RIEKDWYTLMNTIINGSASE--ADAARKQLREELLAIAPVFGQKPYFLSDEFSLVD functional relA gene is required for SspA to regulate gene expression in E. coli. Numerous

I + + + E R QL ++L + + + F++ D studies suggested that the stringent response is characterized by the synthesis of ppGpp,

GstA YIATELHKGFTPLFRPDTPEEYKPTVRAQLEKKLQYVNEALKDEHWICGQRFTIAD

which is catalyzed by relA. Up to date, the molecular basis how ppGpp is required for

SspA CYLAPLL-WRLPQLGIEFSGPGAKELKGYMTRVFERDSFLASLTEAEREMRLGRS SspA function in gene expression is largely unknown. The fluorescence intensity of SspA

YL +L W ++ + G + + +M R+ ER decreased in the presence of ppGpp (Figure 5), which allowed us to determine the

GstA AYLFTVLRW---AYAVKLNLEGLEHIAAFMQRMAERPEVQDALSAEGLK------ dissociation constant.

Figure 1. Forty seven amino acids are identical. The additional positive 35 amino acids

are marked as +. This figure shows that the amino acid sequence of SspA is not much

homologous to GSTs, although their three-dimensional structures are well conserved.

Figure 5. Determination of the dissociation constant (KD) for ppGpp on SspA by

fluorescence spectroscopy. (a) The fluorescence spectra of SspA in the presence of

various concentrations of ppGpp. SspA was excited at 280 nm and the emission spectra

of SspA (10 μg/mL) were measured at 25 ⁰C. (b) The fluorescence changes (ΔF) at 330

nm against ppGpp concentrations.

Figure 2. Structural comparisons of E. coli GstA and SspA. (a) Crystal structure of GstA.

(b) The structure of SspA was generated by SWISS-MODEL(1yy7) and visualized by

PyMol.25 Escherichia coli SspA is highly similar to GstA structurally. The Cys11 and Table 1. Dissociation constants KD of SspA for ppGpp, CDNB, GSH, and

His106 at the active site of GstA are shown in (a) and the corresponding Tyr21 and glutathionesulfonic acid.

Tyr111 are in (b). Three tryptophan residues, responsible for the intrinsic fluorescence

of SspA are also visualized. KD (µM)

Biding of GSH and CDNB to SspA Examined by Fluorescence ppGpp 109.1 ± 7.2

Spectroscopy. CDNB 75.3 ± 4.3

Escherichia coli SspA showed a strong intrinsic fluorescence at 330 nm when excited at GSH 225.0 ± 34.4

280 nm. Three tryptophan residues in SspA may contribute to the fluorescence. The Glutathionesulfonate 272.0 ± 52.1

fluorescence intensity of SspA decreased in the presence of GSH, CDNB, and

glutathionesulfonate (Figures 3 and 4).

(Conclusion)

SspA from E. coli shows a sequence homology to glutathione S-

transferase and acts as a transcription factor. Although SspA does

not have glutathione S-transferase activity, it binds strongly with

both substrates, GSH and CDNB. SspA also binds with ppGpp,

which may provide the molecular basis why relA, the gene for

ppGpp synthetase, is required for gene regulation by SspA. SspA

may act like DksA, a ppGpp-binding transcription factor identified

Figure 3. Determination of the dissociation constant (KD) for GSH on SspA by fluorescen previously.

ce spectroscopy. (a) The fluorescence spectra of SspA in the presence of various concent

rations of GSH. While excited at 280 nm, the emission spectra of SspA (10 μg/mL) were

recorded at 25 ⁰C. (b) The fluorescence changes (ΔF) at 330 nm against GSH concentrati

ons.