Journal of Molecular Biology
Volume 295, Issue 2, 14 January 2000, Pages 225-237
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Regular article
Solution structure and backbone dynamics of the second PDZ domain of postsynaptic density-951

https://doi.org/10.1006/jmbi.1999.3350Get rights and content

Abstract

The second PDZ domain of postsynaptic density-95 (PSD-95 PDZ2) plays a critical role in coupling N-methyl-d-aspartate receptors to neuronal nitric oxide synthase (nNOS). In this work, the solution structure of PSD-95 PDZ2 was determined to high resolution by NMR spectroscopy. The structure of PSD-95 PDZ2 was compared in detail with that of α1-syntrophin PDZ domain, as the PDZ domains share similar target interaction properties. The interaction of the PSD-95 PDZ2 with a carboxyl-terminal peptide derived from a cytoplasmic protein CAPON was studied by NMR titration experiments. Complex formation between PSD-95 PDZ2 and the nNOS PDZ was modelled on the basis of the crystal structure of the α1-syntrophin PDZ/nNOS PDZ dimer. We found that the prolonged loop connecting the βB and βC strands of PSD-95 PDZ2 is likely to play a role in both the binding of the carboxyl-terminal peptide and the nNOS β-finger. Finally, the backbone dynamics of the PSD-95 PDZ2 in the absence of bound peptide were studied using a model-free approach. The “GLGF”-loop and the loop connecting αB and βF of the protein display some degree of flexibility in solution. The rest of the protein is rigid and lacks detectable slow time-scale (microseconds to milliseconds) motions. In particular, the loop connecting βB and βC loop adopts a well-defined, rigid structure in solution. It appears that the loop adopts a pre-aligned conformation for the PDZ domain to interact with its targets.

Introduction

Signal transmission in neurons is mediated by a wide variety of ion channels and receptors specifically localized at the synaptic membrane. Rather than freely diffusing in the membrane, these ion channels and receptors form multimeric clusters (for reviews, see Sheng 1996, Craven and Bredt 1998, Colledge and Froehner 1998, O’Brien et al 1998). Though the molecular mechanisms underlying assembly of the protein networks are largely unknown, recent studies have identified elements critical for synaptic clustering of certain ion channels Sheng 1996, Craven and Bredt 1998, Colledge and Froehner 1998, O’Brien et al 1998. For example, clustering of nicotinic acetylcholine receptors at neuromuscular endplates requires a specific 43 kDa protein rapsyn (Apel et al., 1995). More generally, a large class of PDZ (PSD-95, DLG, and ZO-1) domain-containing proteins were identified that mediate targeting and clustering of channels, receptors, cell adhesion proteins, and other signalling enzymes at the specific sites of cell-cell contact, including synapses Sheng 1996, Kornau et al 1997, Craven and Bredt 1998, Colledge and Froehner 1998, O’Brien et al 1998. The membrane-associated guanylate kinase (MAGUK) proteins is a well-studied family of PDZ domain-containing proteins. Mammalian MAGUK proteins include PSD-95/SAP90 Cho et al 1992, Kistner et al 1993, PSD-93/chapsyn-110 Brenman et al 1996b, Kim et al 1996, SAP-97/hdlg Lue et al 1994, Muller et al 1995, and SAP-102 (Müller et al., 1996), all of which are found at synapses (reviewed by Craven & Bredt, 1998). MAGUKs share common domain organizations consisting of three PDZ domains at the N terminus followed by an SH3 domain. The C-terminal region of MAGUKs encodes a guanylate kinase-like (GK) domain; however, no kinase activity has been detected for the MAGUK proteins. Like the PDZ domains and the SH3 domain, the guanylate kinase domain of MAGUKs functions as a protein-protein interaction module Naisbitt et al 1997, Brenman et al 1998. The SH3 domain and the GK domain of PSD-95 were found to interact with each other, and such interaction may regulate the protein interaction properties of the modules (McGee & Bredt, 1999).

Much of the current knowledge regarding channel clustering and organizing of the signaling complex by the MAGUK proteins has come from studies of PSD-95. Mutation of PSD-95 in mice leads to an enhanced long-term potentiation and impaired learning (Migaud et al., 1998). The protein can multimerize via two Cys residues in the N terminus, and the Cys residues play important roles in targeting PSD-95 to cell membrane Hsueh et al 1997, Craven et al 1999, Hsueh and Sheng 1999. The first two PDZ domains of PSD-95 can bind specifically to the Shaker K+ channel or N-methyl-d-aspartate (NMDA) receptor NR2 subunits via a sequence motif of -E-S/T-X-V∗ located at the extreme C termini Kim et al 1995, Kornau et al 1995, Niethammer et al 1996. The second PDZ domain of PSD-95 also binds to the PDZ domain of neuronal nitric oxide synthase (nNOS) (Brenman et al., 1996a). Suppression of PSD-95 expression by antisense technology attenuates NO production via NMDA receptor-mediated Ca2+ influx, suggesting that PSD-95 specifically couples NMDA receptor activation to NO neurotoxicity (Sattler et al., 1999). The discovery of the specific coupling between the NMDA receptor and nNOS by the second PDZ domain of PSD-95 makes this PDZ domain an attractive target for designing therapeutic drugs against stroke.

Canonical PDZ domains contain ∼80–100 amino acid residues. The PDZ domains form a compact, globular structure consisting of a six-stranded antiparallel β-barrel flanked by two α-helices Doyle et al 1996, Cabral et al 1996. The carboxyl peptide binds to a groove formed by βB and αB (Doyle et al., 1996). Both NMR and X-ray studies showed that the nNOS PDZ domain contains an extra two-strand antiparallel β-sheet C-terminal to the canonical PDZ domain Tochio et al 1999, Hillier et al 1999. It was proposed that this β-sheet extension is likely to bind to the PDZ2 peptide-binding groove of PSD-95 (Tochio et al., 1999). The crystal structure of the nNOS PDZ/α1-syntrophin PDZ dimer indeed showed that the β-sheet extension of the nNOS PDZ binds to the peptide-binding groove formed by βB and αB of α-syntrophin via β-invasion (Hillier et al., 1999).

In this work, we determined the high-resolution solution structure of the second PDZ domain of PSD-95. The interaction of the PDZ domain with a C-terminal peptide was also investigated. The backbone dynamics of the PDZ domain was studied in detail by 15N-relaxation experiments using a model-free approach.

Section snippets

Structural determination

The three-dimensional structure of the second PDZ domain of PSD-95 (referred as PSD-95 PDZ2), that encompasses amino acid residues 155–249 of the native protein was determined in aqueous solution at pH 6.0, 30 °C, using a total of 1835 experimental restraints (Table 1). PSD-95 PDZ2 is a monomeric protein at a concentration up to ∼1.5 mM used for NMR studies. The narrow linewidth of the NMR signals and low degree chemical shift degeneration allowed us to obtain a large number of unambiguous NOEs

Cloning, expression, and purification of PSD-95 PDZ2

The second PDZ domain of rat PSD-95 encompassing amino acid residues 155-249 of the protein was PCR amplified from the full-length PSD-95 gene using the following two primers: 5′-CTGCTCGAGGCCGAAAAG- GTC-3′ (coding strand) and 5′-CTGGATCCTAGGCAT- TGCTG-3′ (non-coding strand). The amplified PSD-95 PDZ2 fragment was inserted into the Xho I and Bam HI sites of the plasmid pET14b (Novagen). The pET14b plasmid harboring the PSD-95 PDZ2 gene was then transformed into Escherichia coli BL21(DE3) host

Acknowledgements

We thank L. E. Kay for providing NMR pulse sequences, A. G. Palmer for the program Modelfree 4.0 for relaxation data analysis, S. R. Jaffrey for the CAPON clone, Dr M. Nilges for the help in torsion angle dynamic calculations of the NMR structures, and D. Miller- Martini for critical reading and comments on the manuscript. This work is partially supported by grants from the Research Grant Council of Hong Kong to M.Z. (HKUST6189/97 M, 6198/99 M). The NMR spectrometers used in this work were

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