NMR STUDIES ON STRUCTURE AND DYNAMICS OF TWO-DOMAIN PROTEINS

The general objective of the present thesis work was to examine 3-dimensional structure as well as the motional properties of two different types of two-domain proteins by solution-state NMR. One of the major tasks of this thesis work was to investigate the domain orientation of these molecules by employing residual dipolar couplings (RDC) which can be used as orientational restraints for increasing the accuracy of protein structures determined by NMR. In the first paper of this thesis two neighboring Epidermal Growth Factor modules (EGF) of Protein S were investigated. Protein S is a down-regulator of blood coagulation and possesses 4 tandem EGF domains. The structure determination of EGF3-4 [Protein Data Bank accession code: 1z65c], employing RDCs, suggests that there is a hinge-like motion in EGF 3, which results in a bending of the structure. This is a unique and so far unprecedented fold of an EGF module. The second paper deals with the structures of human and porcine Beta-Microseminoprotein, MSP, alternatively called Prostatic Secretory Protein of 94 amino acids, PSP94. The structures of the two proteins from these organisms were shown to be very similar [Protein Data Bank accession codes: 2iz3 and 2iz4]. MSP comprises two distinct beta-sheet domains. MSP adopts an extended structure, as determined by employing three different types of RDCs in the structure calculations. This result is in contrast to a recently published structure of porcine MSP (Wang et al. 2005, Journal of Molecular Biology, 346, 1071-1082; Protein Data Bank accession code: 1xhh), where the domains are arranged in a compact conformation. Structure validation showed that the usage of only one type of RDCs cannot solely discriminate between the two different domain orientations. In a subsequent study 15N relaxation data of human and porcine MSP were measured and compared. Model-free analysis of the heteronuclear relaxation data (R1, R2 and steady state NOE of the backbone {1H}-15N) was used to extract information on the dynamics of the proteins. The dynamical information obtained by this method reports on both the overall tumbling and the internal motion of human and porcine MSP. The data show that the human variant of MSP is more flexible than its porcine counterpart. A combination of CPMG and R1,rho experiments was employed to characterize the chemical exchange contribution to R2 for residues of human MSP. It was shown that in human MSP backbone amide nitrogens of at least two residues, Glu31 and Tyr43, experience chemical exchange on a timescale of 0.2 ms. It has recently been shown that human MSP binds to two blood plasma proteins, CRISP-3 ("Cysteine RIch Secretory Protein-3"; Udby et al. 2005, Biochemical and Biophysical Research Communications, 333, 555-561) and PSPBP ("PSP94-Binding Protein"; Reeves et al. 2005, Biochemical Journal, 385, 105-114). Based on the crystal structure of stecrisp, which is a CRISP-3 homologous protein, and NMR data for a 1:1 mixture of human MSP and human CRISP-3, a model for the protein complex is presented in the last paper of this thesis

A model of the complex between human beta-microseminoprotein and CRISP-3 based on NMR data

beta-Microseminoprotein (MSP), a 10kDa seminal plasma protein, forms a tight complex with cysteine-rich secretory protein 3 (CRISP-3) from granulocytes. The 3D structure of human MSP has been determined but there is as yet no 3D structure for CRISP-3. We have now studied the complex between human MSP and CRISP-3 with multidimensional NMR. (15)N-HSQC spectra show substantial differences between free and complexed hMSP. Using several 3D-NMR spectra of triply labeled hMSP in complex with a recombinant N-terminal domain of CRISP-3, most of the backbone of hMSP could be assigned. The data show that only one side of hMSP, comprising beta-strands 1, 4, 5, and 8 are affected by the complex formation, indicating that beta-strands 1 and 8 form the main binding surface. Based on this we present a tentative structure for the hMSP-CRISP-3 complex using the known crystal structure of triflin as a model of CRISP-3

Solution structures of human and porcine beta-microseminoprotein

beta-Microseminoprotein (MSP) is a small cysteine-rich protein (molecular mass about 10 kDa) first isolated from human seminal plasma and later identified in several other organisms. The function of MSP is not known, but a recent study has shown MSP to bind CRISP-3, a protein present in neutrophilic granulocytes. The amino acid sequence is highly variable between species raising the question of the evolutionary conservation of the 3D structure. Here we present NMR solution structures of both the human and the porcine MSP. The two proteins (sequence identity 51%) have a very similar 3D structure with the secondary structure elements well conserved and with most of the amino acid substitutions causing a change of charge localized to one side of the molecule. MSP is a beta-sheet-rich protein with two distinct domains. The N-terminal domain is composed of a four-stranded beta-sheet, with the strands arranged according to the Greek key-motif, and a less structured part. The C-terminal domain contains two two-stranded beta-sheets with no resemblance to known structural motifs. The two domains, connected to each other by the peptide backbone, one disulfide bond, and interactions between the N and C termini, are oriented to give the molecule a rather extended structure. This global fold differs markedly from that of a previously published structure for porcine MSP, in which the two domains have an entirely different orientation to each other. The difference probably stems from a misinterpretation of ten specific inter-domain NOEs. (c) 2006 Elsevier Ltd. All rights reserved

Assessment of the higher order structure of Humira® Remicade® Avastin® Rituxan® Herceptin® and Enbrel® by 2D-NMR fingerprinting

The advent of monoclonal antibody biosimilar products has stimulated the development of analytical methods that can better characterize an important quality attribute, namely the higher order structure (HOS). Here, we propose a simple approach based on heteronuclear 2D NMR techniques at natural abundance for generating spectral fingerprints of the HOS at high resolution. We show that the proposed method can assess the HOS of six therapeutic products, adalimumab (Humira®), bevacizumab (Avastin®), infliximab (Remicade®), rituximab (Rituxan®), trastuzumab (Herceptin®), and Etanercept (Enbrel®). After treatment with immobilized papain, the purified fragments (Fab and Fc) were analyzed by 2D proton-nitrogen and proton-carbon NMR correlations. All Fab and Fc fragments produced high-resolution 2D-NMR spectra from which assessment of their higher order structure can be performed in the context of comparability studies. In particular, the two different sequences of Fc fragments could be unambiguously distinguished. The results show that it is possible to obtain structurally dependent information at amino acid resolution of these important therapeutic agents

Solution Structure of the Ca(2+)-Binding EGF3-4 Pair from Vitamin K-Dependent Protein S: Identification of an Unusual Fold in EGF3(,).

Vitamin K-dependent protein S is a cofactor of activated protein C, a serine protease that regulates blood coagulation. Deficiency of protein S can cause venous thrombosis. Protein S has four EGF domains in tandem; domains 2-4 bind calcium with high affinity whereas domains 1-2 mediate interaction with activated protein C. We have now solved the solution structure of the EGF3-4 fragment of protein S. The linker between the two domains is similar to what has been observed in other calcium-binding EGF domains where it provides an extended conformation. Interestingly, a disagreement between NOE and RDC data revealed a conformational heterogeneity within EGF3 due to a hinge-like motion around Glu186 in the Cys-Glu-Cys sequence, the only point in the domain where flexibility is allowed. The dominant, bent conformation of EGF3 in the pair has no precedent among calcium-binding EGF domains. It is characterized by a change in the angle of Glu186 from 160 ± 40, as seen in ten other EGF domains, to 0 ± 15. NOESY data suggest that Tyr193, a residue not conserved in other calcium-binding EGF domains (except in the homologue Gas6), induces the unique fold of EGF3. However, SAXS data, obtained on EGF1-4 and EGF2-4, showed a dominant, extended conformation in these fragments. This may be due to a counterproductive domain-domain interaction between EGF2 and EGF4 if EGF3 is in a bent conformation. We speculate that the ability of EGF3 to adopt different conformations may be of functional significance in protein-protein interactions involving protein S

Activity-Based Protein Profiling of the <i>Escherichia coli</i> GlpG Rhomboid Protein Delineates the Catalytic Core

Rhomboid proteins comprise the largest class of intramembrane protease known, being conserved from bacteria to humans. The functional status of these proteases is typically assessed through direct or indirect detection of peptide cleavage products. Although these assays can report on the ability of a rhomboid to catalyze peptide bond cleavage, differences in measured hydrolysis rates can reflect changes in the structure and activity of catalytic residues, as well as the ability of the substrate to access the active site. Here we show that a highly reactive and sterically unencumbered fluorophosphonate activity-based protein profiling probe can be used to report on the catalytic integrity of active site residues in the <i>Escherichia coli</i> GlpG protein. We used results obtained with this probe on GlpG in proteomic samples, in combination with a conventional assay of proteolytic function on purified samples, to identify residues that are located on the cytoplasmic side of the lipid bilayer that are required for maximal proteolytic activity. Regions tested include the 90-residue aqueous-exposed N-terminus that encompasses a globular structure that we have determined by solution nuclear magnetic resonance, along with residues on the cytoplasmic side of the transmembrane domain core. While in most cases mutation or elimination of these residues did not significantly alter the catalytic status of the GlpG active site, the lipid-facing residue Arg227 was found to be important for maintaining a catalytically competent active site. In addition, we found a functionally critical region outside the transmembrane domain (TMD) core that is required for maximal protease activity. This region encompasses an additional 8–10 residues on the N-terminal side of the TMD core that precedes the first transmembrane segment and was not previously known to play a role in rhomboid function. These findings highlight the utility of the activity-based protein profiling approach for the characterization of rhomboid function

Micelle-Catalyzed Domain Swapping in the GlpG Rhomboid Protease Cytoplasmic Domain

Three-dimensional domain swapping is a mode of self-interaction that can give rise to altered functional states and has been identified as the trigger event in some protein deposition diseases, yet rates of interconversion between oligomeric states are usually slow, with the requirement for transient disruption of an extensive network of interactions giving rise to a large kinetic barrier. Here we demonstrate that the cytoplasmic domain of the <i>Escherichia coli</i> GlpG rhomboid protease undergoes slow dimerization via domain swapping and that micromolar concentrations of micelles can be used to enhance monomer–dimer exchange rates by more than 1000-fold. Detergents bearing a phosphocholine headgroup are shown to be true catalysts, with hexadecylphosphocholine reducing the 26 kcal/mol free energy barrier by >11 kcal/mol while preserving the 5 kcal/mol difference between monomer and dimer states. Catalysis involves the formation of a micelle-bound intermediate with a partially unfolded structure that is primed for domain swapping. Taken together, these results are the first to demonstrate true catalysis for domain swapping, by using micelles that work in a chaperonin-like fashion to unfold a kinetically trapped state and allow access to the domain-swapped form