NMR spectroscopy has been used to characterize the protein-protein interactions between the mouse Grb2 (mGrb2) N-terminal SH3 domain complexed with a 15-residue peptide (SPLLPKLPP-KTYKRE) corresponding to residues 1264-1278 of the mouse Sos-2 (mSos-2) protein. Intermolecular interactions between the peptide and 13C-15N-labeled SH3 domain were identified in half-reverse-filtered 2D and 3D NOESY experiments. Assignments for the protons involved in interactions between the peptide and the SH3 domain were confirmed in a series of NOESY experiments using a set of peptides in which different leucine positions were fully deuterated. The peptide ligand-binding site of the mGrb2 N-terminal SH3 domain is defined by the side chains of specific aromatic residues (Tyr7, Phe9, Trp36, Tyr52) that form two hydrophobic subsites contacting the side chains of the peptide Leu4 and Leu7 residues. An adjacent negatively charged subsite on the SH3 surface is likely to interact with the side chain of a basic residue at peptide position 10 that we show to be involved in binding. The peptide-binding site of the SH3 is characterized by large perturbations of amide chemical shifts when the peptide is added to the SH3 domain. The mGrb2 N-terminal SH3 domain structure in the complex is well-defined (backbone RMSD of 0.56 +/- 0.21 calculated over the backbone N, C alpha, and C atoms of residues 1-54). The structure of the peptide in the complex is less well-defined but displays a distinct orientation.(ABSTRACT TRUNCATED AT 250 WORDS)

Human cytomegalovirus (HCMV) encodes a protease that cleaves itself and the HCMV assembly protein. Two proteolytic processing sites within the protease were identified at Ala 256-Ser 257 (release site) and Ala 643-Ser 644 (maturation site). Identification of rP5-P4' and mP4-P6' as the minimal peptide substrates spanning the release and maturation cleavage sites, respectively, demonstrated a requirement for residues flanking the conserved core in substrate recognition and hydrolysis, which are unique to HCMV. Kinetic parameters determined for release-site-derived and maturation-site-derived peptides revealed a 10-fold increase in k,,JK,, for a maturational peptide (mP4-PS') over release-site peptide (rP5-P5'). Experimental results with a panel of class-specific protease inhibitors were consistent with the protease being a member of the serine or cysteine family of proteases. Further investigation revealed that the HCMV protease activity decreased with incorporation of ['4C]iodoacetic acid, but when approximately 4.5 mol I4C were incorporatedmol enzyme, the enzyme retained approximately 20% of its original activity, indicating that hydrolysis does not require a cysteine nucleophile. Analysis of diisopropyl-fluorophosphate-inactivated protease by mass spectrometry indicated a stoichiometry of 1 diisopropyl phosphate/protease molecule, suggesting that hydrolysis requires a single serine nucleophile. The residue modified by diisopropyl fluorophosphate was identified as Ser132 by modification with 'H-labeled diisopropyl fluorophosphate, peptide mapping and Edman degradation. This residue and the region in which it is found is highly conserved among the herpes virus proteases. These data demonstrates that HCMV protease is a serine protease and that Ser132 is the active-site nucleophile.Members of the herpes virus family, including human cytomegalovirus (HCMV) and HSV-1, encode an assembly protein which is a major component of intermediate B-capsids. The assembly protein is only transiently associated with virus particles, and is absent from mature virions [l -31. As a consequence, the assembly protein is proposed to play a role in virus maturation analogous to that of the scaffolding protein of bacteriophages [4]. During infection, the HCMV preassembly protein undergoes proteolytic processing to form the assembly protein, a lower molecular-mass species that lacks 64 amino acids at the carboxy terminus [5]. Mutants of HSV-1, defective in processing the HSV-1 assembly protein, form aberrant empty capsids and fail to package DNA, indicating that proteolytic processing of the virus assembly protein is critical for herpes virus particle maturation [6, 71.The protease responsible for processing the preassembly protein during HCMV infection consists of 708 amino acids Abbreviations. HCMV, human cytomegalovirus; iPr,PF, diisopropylfluorophosphate; ESI, electrospray ionization; GST, glutathione S-transferase; HSV-1. herpes simplex virus type 1; SCMV, simian cytomegalovirus. encoded by the UL80 open reading frame, which is 3' co...

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