NMR Amide Research Articles

Conformational dynamics in interleukin 17A and 17F functional complexes is a key determinant of receptor A affinity and specificity

The proinflammatory cytokines IL-17A and IL-17F have been identified as key drivers of a range of human inflammatory diseases, such as psoriasis, which has led to several therapeutic antibodies targeted at IL-17A. The two cytokines have been shown to tightly associate as functional homo and hetero dimers, which induce signalling via the formation of a cell surface signalling complex with a single copy of both IL-17RA and IL-17RC. Striking differences in affinity have been observed for IL-17RA binding to IL-17AA, IL-17AF and IL-17FF, however, the functional significance and molecular basis for this has remained unclear. We have obtained comprehensive backbone NMR assignments for full length IL-17AA (79%), IL-17AF (93%) and IL-17FF (89%), which show that the dimers adopt almost identical backbone topologies in solution to those observed in reported crystal structures. Analysis of the line widths and intensities of assigned backbone amide NMR signals has revealed striking differences in the conformational plasticity and dynamics of IL-17AA compared to both IL-17AF and IL-17FF. Our NMR data indicate that a number of regions of IL-17AA are interconverting between at least two distinct conformations on a relatively slow timescale. Such conformational heterogeneity has previously been shown to play an important role in the formation of many high affinity protein-protein complexes. The locations of the affected IL-17AA residues essentially coincides with the regions of both IL-17A and IL-17F previously shown to undergo significant structural changes on binding to IL-17RA. Substantially less conformational exchange was revealed by the NMR data for IL-17FF and IL-17AF. We propose that the markedly different conformational dynamic properties of the distinct functional IL-17 dimers plays a key role in determining their affinities for IL-17RA, with the more dynamic and plastic nature of IL-17AA contributing to the significantly tighter affinity observed for binding to IL-17RA. In contrast, the dynamic properties are expected to have little influence on the affinity of IL-17 dimers for IL-17RC, which has recently been shown to induce only small structural changes in IL-17FF upon binding.

In Vitro Production of Perdeuterated Proteins in H2O for Biomolecular NMR Studies.

The cell-free synthesis is an efficient strategy to produce in large scale protein samples for structural investigations. In vitro synthesis allows for significant reduction of production time, simplification of purification steps and enables production of both soluble and membrane proteins. The cell-free reaction is an open system and can be performed in presence of many additives such as cofactors, inhibitors, redox systems, chaperones, detergents, lipids, nanodisks, and surfactants to allow for the expression of toxic membraneproteins or intrinsically disordered proteins. In this chapter we present protocols to prepare E. coli S30 cellular extracts, T7 RNA polymerase, and their use for in vitro protein expression. Optimizations of the protocol are presented for preparation of protein samples enriched in deuterium, a prerequisite for the study of high-molecular-weight proteins by NMR spectroscopy. An efficient production of perdeuterated proteins is achieved together with a full protonation of all the amide NMR probes, without suffering from residual protonation on aliphatic carbons. Application to the production of the 468kDa TET2 protein assembly for NMR investigations is presented.

Structural dynamics of a spinlabeled ribosome elongation factor P (EF-P) from Staphylococcus aureus by EPR spectroscopy

Elongation factor P (EF-P) is a three domain protein that binds to the ribosome between P and E sites.The EF-P involved in a specialized translation of stalling amino acid motifs such as (PPP or APP). Proteins with stalling motifs are involved in various cell processes, including stress resistance and virulence of bacteria. EF-P stabilizes the P-tRNA and increases the entropy of stalled ribosome complex to compensate for rigid nature of proline residue, thus providing adequate protein synthesis rate. Detailed structural mechanisms of this effect are still poorly understood. It was shown that most of bacteria needs a special post-translational modification in a conservative region of the loop in the domain I of the EF-P located near the CCA-end of P-tRNA and the peptidyl transferase center. In present paper by EPR spectroscopy we investigated a spinlabeled EF-P from Staphylococcus aureus—a pathogenic bacteria which causes various human diseases. Addition of MTSL ((S-(1-oxyl-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrol-3-yl)methyl methanesulfonothioate)) label covalently bound to 32Cys in the highly conservative part of EF-P loop in the domain I of the EF-P allows us to analyze protein dynamic by EPR spectroscopy of the high mobility region which was not observed in NMR spectra due to fast proton exchange leading to the absence of the corresponding amide NMR resonances. We suppose that our result could be extended in future for the analysis of EF-P—ribosome complex formation process by advanced EPR techniques.

Charged cyclic hexapeptides: Updating molecular descriptors for permeability purposes

Three polar cyclic hexapeptides differently charged at physiological pH (1 = neutral, 2 = anionic, 3 = cationic) were synthesized and their cell permeability measured. Lipophilicity in octanol/water didn't account for the higher permeability of the cationic derivative but three chromatographic indexes (log KwIAM, log k′ HILIC and log k′ c-HILIC) were more efficient to this respect. NMR amide chemical shift temperature coefficients (ΔδNH/ΔT) were used to explore the IMHB network of the backbone. MD simulations in different environments (water, chloroform and DMPC lipid bilayer) highlighted that the charged amino group of the lysine moiety of 3 is not involved in the formation of any IMHB in water whereas a different behavior is registered in chloroform and DMPC lipid bilayer. Overall this paper highlights how a combination of experimental and computational approaches could help in comparing permeability and physicochemical properties of neutral and charged cyclic peptides.

Rational design and synthesis of an orally bioavailable peptide guided by NMR amide temperature coefficients.

Enhancing the oral bioavailability of peptide drug leads is a major challenge in drug design. As such, methods to address this challenge are highly sought after by the pharmaceutical industry. Here, we propose a strategy to identify appropriate amides for N-methylation using temperature coefficients measured by NMR to identify exposed amides in cyclic peptides. N-methylation effectively caps these amides, modifying the overall solvation properties of the peptides and making them more membrane permeable. The approach for identifying sites for N-methylation is a rapid alternative to the elucidation of 3D structures of peptide drug leads, which has been a commonly used structure-guided approach in the past. Five leucine-rich peptide scaffolds are reported with selectively designed N-methylated derivatives. In vitro membrane permeability was assessed by parallel artificial membrane permeability assay and Caco-2 assay. The most promising N-methylated peptide was then tested in vivo. Here we report a novel peptide (15), which displayed an oral bioavailability of 33% in a rat model, thus validating the design approach. We show that this approach can also be used to explain the notable increase in oral bioavailability of a somatostatin analog.

Capping of the N-terminus of PSD-95 by calmodulin triggers its postsynaptic release

Postsynaptic density protein-95 (PSD-95) is a central element of the postsynaptic architecture of glutamatergic synapses. PSD-95 mediates postsynaptic localization of AMPA receptors and NMDA receptors and plays an important role in synaptic plasticity. PSD-95 is released from postsynaptic membranes in response to Ca(2+) influx via NMDA receptors. Here, we show that Ca(2+)/calmodulin (CaM) binds at the N-terminus of PSD-95. Our NMR structure reveals that both lobes of CaM collapse onto a helical structure of PSD-95 formed at its N-terminus (residues 1-16). This N-terminal capping of PSD-95 by CaM blocks palmitoylation of C3 and C5, which is required for postsynaptic PSD-95 targeting and the binding of CDKL5, a kinase important for synapse stability. CaM forms extensive hydrophobic contacts with Y12 of PSD-95. The PSD-95 mutant Y12E strongly impairs binding to CaM and Ca(2+)-induced release of PSD-95 from the postsynaptic membrane in dendritic spines. Our data indicate that CaM binding to PSD-95 serves to block palmitoylation of PSD-95, which in turn promotes Ca(2+)-induced dissociation of PSD-95 from the postsynaptic membrane.

A simplified recipe for assigning amide NMR signals using combinatorial 14N amino acid inverse-labeling

Assignment of backbone amide proton resonances is one of the most time-consuming stages of any protein NMR study when the protein samples behave non-ideally. A robust and convenient NMR procedure for analyzing spectra of marginal-to-low quality is helpful for high-throughput structure determination. The 14N selective- and inverse-labeling method is a candidate solution. Here, we present a simplified protocol for assigning protein backbone amide NMR signals. When 14N inversely labeled residues are present in a protein, their backbone NH cross peaks vanish from the protein's 1H-(15)N HSQC spectrum, and thus, their chemical shifts can be readily identified by a process of elimination. Some metabolically related amino acids, for example, Ile, Leu, and Val, cannot be individually incorporated but can be inversely labeled together. We optimized and simplified the protocol and M9-based medium formula for the 14N selective- and inverse-labeling method without any additives. Our approach should be cost-effective, because the method could be additively applied stepwise, even when the proteins of interest were found to be non-ideal.

Solid-Phase Synthesis and NMR Structural Studies of the Marine Antibacterial Cyclic Tetrapeptide: Cyclo[GSPE

Twelve-membered head-to-tail cyclic tetrapeptides (CTPs) are rigid molecules found in nature and possess a diverse range of biological activities. A possible reason may be due to their ability to adopt rigid conformations in solution mimicking reverse-turns. Reverse-turns are common structural motifs which serve as molecular recognition sites in many protein-receptor interactions. In this paper, we describe the solid-phase synthesis of the antibacterial cyclic tetrapeptide cyclo[Gly-Ser-Pro-Glu] (cyclo[GSPE]), first isolated from the Ruegeria strain of marine bacteria by Mitova et al. (J Nat Prod 67:1178–1181, 2004). Our NMR experiments in H2O:D2O:DMSO (18:1:1) revealed that it possessed three conformations in an approximate ratio of 4:2:1 based on NMR amide peak intensities. 2D NMR studies and computer calculations revealed that the major conformer adopted a reverse-turn conformation and have ω torsion angles twisted by up to 2°, with two transoid amide bonds between Gly-Ser, Pro-Glu and two cisoid amide bonds between Ser-Pro, Glu-Gly in a cis–trans-cis–trans (ctct) pattern. This supports previous reports that majority of CTPs adopt a ctct pattern when dissolved in hydrogen-bond disrupting solvents (Che and Marshall in J Med Chem 49:111–124, 2006 and references cited therein). An ensemble of ten lowest-energy-minimised 3D structures generated using XPLOR-NIH software revealed that cyclo[GSPE] possessed a rigid backbone ring scaffold. The remaining two minor conformers were present in quantities too low for NMR structural studies.

Steering Protein−Ligand Docking with Quantitative NMR Chemical Shift Perturbations

Lead optimization benefits from including structural knowledge of the target. We present a new method that exploits quantitatively NMR amide proton chemical shift perturbations (CSP) on the protein side for protein-ligand docking. The approach is based on a hybrid scoring scheme consisting of a weighted sum of DrugScore, describing protein-ligand interactions, and Kendall's rank correlation coefficient, which scores ligand poses with respect to their agreement with experimental CSP data. For back-calculating CSP for a ligand pose, an efficient empirical model considering only ring-current effects is applied. The hybrid scoring scheme has been implemented in AutoDock. Compared to previous approaches, the rank correlation provides a measure that is more robust against the presence of outliers in back-calculated CSP data. Furthermore, our methods exploit CSP information at docking time and not for postfiltering, resulting in an enhanced generation of native-like solutions. As we exploit CSP information quantitatively, the experimental information effectively contributes to orient the ligand in the binding site. When applied to 70 protein-ligand complexes with computed CSP reference data, the docking success rate increases from 71%, if no CSP information is used, to 99% at the highest CSP weighting factor tested. Global optimization, thus, performs satisfactorily on the hybrid docking energy landscape. We next applied the approach to three test cases with experimental CSP reference data. Without CSP information, neither of the complexes is successfully docked. Including CSP information with the same CSP weighting factor, as determined above, leads to successful docking in all three cases. Only then native-like ligand configurations are generated at two of the three complexes. Binding site movements of up to 2 A are found to not deteriorate the docking success. The approach will be particularly important for protein-ligand complexes that are difficult to predict computationally, such as ligands binding to flat interface regions.

Scanning Mutagenesis of α-Conotoxin Vc1.1 Reveals Residues Crucial for Activity at the α9α10 Nicotinic Acetylcholine Receptor

Vc1.1 is a disulfide-rich peptide inhibitor of nicotinic acetylcholine receptors that has stimulated considerable interest in these receptors as potential therapeutic targets for the treatment of neuropathic pain. Here we present an extensive series of mutational studies in which all residues except the conserved cysteines were mutated separately to Ala, Asp, or Lys. The effect on acetylcholine (ACh)-evoked membrane currents at the alpha9alpha10 nicotinic acetylcholine receptor (nAChR), which has been implicated as a target in the alleviation of neuropathic pain, was then observed. The analogs were characterized by NMR spectroscopy to determine the effects of mutations on structure. The structural fold was found to be preserved in all peptides except where Pro was substituted. Electrophysiological studies showed that the key residues for functional activity are Asp(5)-Arg(7) and Asp(11)-Ile(15), because changes at these positions resulted in the loss of activity at the alpha9alpha10 nAChR. Interestingly, the S4K and N9A analogs were more potent than Vc1.1 itself. A second generation of mutants was synthesized, namely N9G, N9I, N9L, S4R, and S4K+N9A, all of which were more potent than Vc1.1 at both the rat alpha9alpha10 and the human alpha9/rat alpha10 hybrid receptor, providing a mechanistic insight into the key residues involved in eliciting the biological function of Vc1.1. The most potent analogs were also tested at the alpha3beta2, alpha3beta4, and alpha7 nAChR subtypes to determine their selectivity. All mutants tested were most selective for the alpha9alpha10 nAChR. These findings provide valuable insight into the interaction of Vc1.1 with the alpha9alpha10 nAChR subtype and will help in the further development of analogs of Vc1.1 as analgesic drugs.

The Denatured State of N-PGK Is Compact and Predominantly Disordered

The organisation of the structure present in the chemically denatured N-terminal domain of phosphoglycerate kinase (N-PGK) has been determined by paramagnetic relaxation enhancements (PREs) to define the conformational landscape accessible to the domain. Below 2.0 M guanidine hydrochloride (GuHCl), a species of N-PGK (denoted I b) is detected, distinct from those previously characterised by kinetic experiments [folded (F), kinetic intermediate (I k) and denatured (D)]. The transition to I b is never completed at equilibrium, because F predominates below 1.0 M GuHCl. Therefore, the ability of PREs to report on transient or low population species has been exploited to characterise I b. Five single cysteine variants of N-PGK were labelled with the nitroxide electron spin-label MTSL [(1-oxyl-2,2,5,5-tetramethyl-3-pyrroline-3-methyl)methanesulfonate] and the denaturant dependences of the relaxation properties of the amide NMR signals between 1.2 and 3.6 M GuHCl were determined. Significant PREs for I b were obtained, but these were distributed almost uniformly throughout the sequence. Furthermore, the PREs indicate that no specific short tertiary contacts persist. The data indicate a collapsed state with no coherent three-dimensional structure, but with a restricted radius beyond which the protein chain rarely reaches. The NMR characteristics of I b indicate that it forms from the fully denatured state within 100 μs, and therefore a rapid collapse is the initial stage of folding of N-PGK from its chemically denatured state. By extrapolation, I b is the predominant form of the denatured state under native conditions, and the non-specifically collapsed structure implies that many non-native contacts and chain reversals form early in protein folding and must be broken prior to attaining the native state topology.

MMP-12 Catalytic Domain Recognizes Triple Helical Peptide Models of Collagen V with Exosites and High Activity

Matrix metalloproteinase (MMP)-12 (or metalloelastase) efficiently hydrolyzed the gelatinase-selective alpha1(V)436-447 fluorescent triple helical peptide (THP) when the substrate was submicromolar. The sequence of this THP was derived from collagen V, a component of collagen I fibrils. The hemopexin domains of MMP-12 and -9 each increased k(cat)/K(m) toward this substrate by decreasing K(m), just as the hemopexin domain of MMP-1 enhances its triple helical peptidase activity. Non-fluorescent alpha1(V) THP subtly perturbed amide NMR chemical shifts of MMP-12 not only in the active site cleft but also at remote sites of the beta-sheet and adjoining loops. The alpha1(V) THP protected MMP-12 from the NMR line broadening effects of Gd .EDTA in the active site cleft and more dramatically in the V-B loop next to the primed subsites. Mutagenesis of the exosite in the V-B loop at Thr-205 and His-206 that vary among MMP sequences established that this site supports the high specific activity toward alpha1(V) fluorescent THP without affecting general MMP activity. Surprisingly the alpha1(V) THP also protected novel surfaces in the S-shaped metal-binding loop and beta-strands III and V that together form a pocket on the remote side of the zinc binding site. The patterns of protection suggest bending of the triple helical peptide partly around the catalytic domain to reach novel exosites. Partial unwinding or underwinding of the triple helix could accompany this to facilitate its hydrolysis.

NMR Solution Structures of the Apo and Peptide-Inhibited Human Rhinovirus 3C Protease (Serotype 14): Structural and Dynamic Comparison

The human rhinovirus (HRV) is a positive sense RNA virus responsible for about 30% of "common colds". It relies on a 182 residue cysteine protease (3C) to proteolytically process its single gene product. Inhibition of this enzyme in vitro and in vivo has consistently demonstrated cessation of viral replication. This suggests that 3C protease inhibitors could serve as good drug candidates. However, significant proteolytic substrate diversity exists within the 110+ known rhinovirus serotypes. To investigate this variability we used NMR to solve the structure of the rhinovirus serotype 14 3C protease (subgenus B) covalently bound to a peptide (acetyl-LEALFQ-ethylpropionate) inhibitor. The inhibitor-bound structure was determined to an overall rmsd of 0.82 A (backbone atoms) and 1.49 A (all heavy atoms). Comparison with the X-ray structure of the serotype 2 HRV 3C protease from subgenus A (51% sequence identity) bound to the inhibitor ruprintrivir allowed the identification of conserved intermolecular interactions involved in proximal substrate binding as well as subgenus differences that might account for the variability observed in SAR studies. To better characterize the 3C protease and investigate the structural and dynamic differences between the apo and bound states we also solved the solution structure of the apo form. The apo structure has an overall rmsd of 1.07 +/- 0.17 A over backbone atoms, which is greater by 0.25 A than what is seen for the inhibited enzyme (2B0F.pdb). This increase is localized to the enzyme's C-terminal beta-barrel domain, which is responsible for recognizing and binding proteolytic substrates. Amide hydrogen exchange dynamics revealed dramatic differences between the two enzyme states. Furthermore, a number of residues exhibited exchange-broadened amide NMR signals in the apo state compared to the inhibited state. The majority of these residues are associated with proteolytic substrate interaction.

Carboxyl pKa Values, Ion Pairs, Hydrogen Bonding, and the pH-dependence of Folding the Hyperthermophile Proteins Sac7d and Sso7d

Sac7d and Sso7d are homologous, hyperthermophile proteins with a high density of charged surface residues and potential ion pairs. To determine the relative importance of specific amino acid side-chains in defining the stability and function of these Archaeal chromatin proteins, pKa values were measured for the acidic residues in both proteins using 13C NMR chemical shifts. The stability of Sso7d enabled titrations to pH 1 under low-salt conditions. Two aspartate residues in Sso7d (D16 and D35) and a single glutamate residue (G54) showed significantly perturbed pKa values in low salt, indicating that the observed pH-dependence of stability was primarily due to these three residues. The pH-dependence of backbone amide NMR resonances demonstrated that perturbation of all three pKa values was primarily the result of side-chain to backbone amide hydrogen bonds. Few of the significantly perturbed acidic pKa values in Sac7d and Sso7d could be attributed to primarily ion pair or electrostatic interactions. A smaller perturbation of E48 (E47 in Sac7d) was ascribed to an ion pair interaction that may be important in defining the DNA binding surface. The small number (three) of significantly altered pKa values was in good agreement with a linkage analysis of the temperature, pH, and salt-dependence of folding. The linkage of the ionization of two or more side-chains to protein folding led to apparent cooperativity in the pH-dependence of folding, although each group titrated independently with a Hill coefficient near unity. These results demonstrate that the acid pH-dependence of protein stability in these hyperthermophile proteins is due to independent titration of acidic residues with pKa values perturbed primarily by hydrogen bonding of the side-chain to the backbone. This work demonstrates the need for caution in using structural data alone to argue the importance of ion pairs in stabilizing hyperthermophile proteins.

Solution Structure, Divalent Metal and DNA Binding of the Endonuclease Domain from the Replication Initiation Protein from Porcine Circovirus 2

Circoviruses are the smallest circular single-stranded DNA viruses able to replicate in mammalian cells. Essential to their replication is the replication initiator, or Rep protein that initiates the rolling circle replication (RCR) of the viral genome. Here we report the NMR solution three-dimensional structure of the endonuclease domain from the Rep protein of porcine circovirus type 2 (PCV2), the causative agent of postweaning multisystemic wasting syndrome in swine. The domain comprises residues 12–112 of the full-length protein and exhibits the fold described previously for the Rep protein of the representative geminivirus tomato yellow leaf curl Sardinia virus. The structure, however, differs significantly in some secondary structure elements that decorate the central five-stranded β-sheet, including the replacement of a β-hairpin by an α-helix in PCV2 Rep. The identification of the divalent metal binding site was accomplished by following the paramagnetic broadening of NMR amide signals upon Mn2+ titration. The site comprises three conserved acidic residues on the exposed face of the central β-sheet. For the 1:1 complex of the PCV2 Rep nuclease domain with a 22mer double-stranded DNA oligonucleotide chemical shift mapping allowed the identification of the DNA binding site on the protein and aided in constructing a model of the protein/DNA complex.

Partially Unfolded Forms and Non-two-state Folding of a β-Sandwich: FHA Domain from Arabidopsis Receptor Kinase-associated Protein Phosphatase

FHA domains adopt a β-sandwich fold with 11 strands. The first evidence of partially unfolded forms of a β-sandwich is derived from native-state hydrogen exchange (NHX) of the forkhead-associated (FHA) domain from kinase-associated protein phosphatase from Arabidopsis. The folding kinetics of this FHA domain indicate that EX2 behavior prevails at pH 6.3. In the chevron plot, rollover in the folding arm and bends in the unfolding arm suggest folding intermediates. NHX of this FHA domain suggests a core of six most stable β-strands and two loops, characterized by rare global unfolding events. Flanking this stable core are β-strands and recognition loops with less stability, termed subglobal motifs. These suggest partially unfolded forms (near-native intermediates) with two levels of stability. The spatial separation of the subglobal motifs on the flanks suggests possible parallelism in their folding as additional β-strands align with the stable core of six strands. Intermediates may contribute to differences in stabilities and m-values suggested by NHX or kinetics relative to chemical denaturation. Residual structure in the unfolded regime is suggested by superprotection of β-strand 6 and by GdmCl-dependence of adjustments in amide NMR spectra and residual optical signal. The global folding stability depends strongly on pH, with at least 3 kcal/mol more stability at pH 7.3 than at pH 6.3. This FHA domain is hypothesized to fold progressively with initial hydrophobic collapse of its stable six-stranded core followed by addition of less stable flanking β-strands and ordering of recognition loops.

Cell Migration and Signaling Specificity Is Determined by the Phosphatidylserine Recognition Motif of Rac1

The Rho guanosine triphosphatases (GTPases) control cell shape and motility and are frequently overexpressed during malignant growth. These proteins act as molecular switches cycling between active GTP- and inactive GDP-bound forms. Despite being membrane anchored via their isoprenylated C termini, Rho GTPases rapidly translocate between membrane and cytosolic compartments. Here, we show that the Rho GTPase Rac1 preferentially interacts with phosphatidylserine (PS)-containing bilayers through its polybasic motif (PBM). Rac1 isoprenylation contributes to membrane avidity but is not critical for PS recognition. The similar protein Cdc42 (cell division cycle 42), however, only associates with PS when prenylated. Conversely, other Rho GTPases such as Rac2, Rac3, and RhoA do not bind to PS even when they are prenylated. Cell stimulation with PS induces translocation of Rac1 toward the plasma membrane and stimulates GTP loading, membrane ruffling, and filopodia formation. This stimulation also promotes Cdc42 activation and phosphorylation of mitogen-activated protein kinase through Rac1/PS signaling. Consequently, the PBM specifically directs Rac1 to effect cytoskeletal rearrangement and cell migration by selective membrane phospholipid targeting.

Hsc70 Contacts Helix III of DnaJ‐like Domain from Polyomavirus T Antigens

The J domain of large T antigen, Hsc70, and its hydrolysis of ATP were found necessary for polyomavirus infection to release E2Fs from pRb family members. Hsc70's expected binding site on helix II of the DnaJ-like domain of T antigens is blocked in the crystal structure of a T antigen fragment bound to tumor suppressor pRb. We used NMR to find recognition sites for mammalian Hsc70 upon the J domain of murine polyomavirus T antigens (PyJ). The Hsc70 ATPase domain unexpectedly shifts most of the amide NMR peaks of the C-terminal end of helix III of PyJ. The Hsc70 ATPase domain buries the C-terminal end of helix III of PyJ, protecting it from an uncharged paramagnetic probe. This region corresponds to the QKRAA motif of orthodox J domains. Evolutionary trace analysis suggests helix III of J domains to be of wide importance to specificity for hsp70s. The NMR-detected Hsc70 contacts map to a PyJ surface homologous to a solvent-accessible surface of the SV40 T antigen fragment bound to pRb. This surface of helix III and the HPD loop may allow Hsc70 to form a ternary complex with T antigen and pRb, avoiding obstructions to helix II. The helix III docking site is consistent with large T antigen being able to free an E2F from pRb by positioning the substrate-binding domain of Hsc70 for direct contact with the E2F. NMR spectra show the pRb-binding region of E2F1 (aa243–437) to be unfolded in the absence of DP1. Hsc70 is known to bind unfolded proteins. In the absence of DP1, E2F1(aa243–437) acts as a substrate for Hsc70 in vitro, stimulating its ATPase activity in a manner dependent on PyJ from T antigen. If segments of an E2F are unstructured in vivo, it is conceivable that Hsc70, recruited by large T antigen to a complex with pRb, might act on the unstructured portion of the E2F when liberating it from the pRb family member.

Topological Determinants of Protein Domain Swapping

Protein domain swapping has been repeatedly observed in a variety of proteins and is believed to result from destabilization due to mutations or changes in environment. Based on results from our studies and others, we propose that structures of the domain-swapped proteins are mainly determined by their native topologies. We performed molecular dynamics simulations of seven different proteins, known to undergo domain swapping experimentally, under mildly denaturing conditions and found in all cases that the domain-swapped structures can be recapitulated by using protein topology in a simple protein model. Our studies further indicated that, in many cases, domain swapping occurs at positions around which the protein tends to unfold prior to complete unfolding. This, in turn, enabled prediction of protein structural elements that are responsible for domain swapping. In particular, two distinct domain-swapped dimer conformations of the focal adhesion targeting domain of focal adhesion kinase were predicted computationally and were supported experimentally by data obtained from NMR analyses.

Domain Architectures and Characterization of an RNA-binding Protein, TLS

Translocated in liposarcoma (TLS) is an important protein component of the heterogeneous nuclear ribonucleoprotein complex involved in the splicing of pre-mRNA and the export of fully processed mRNA to the cytoplasm. We examined the domain organization of human TLS by a combined approach using limited proteolysis, matrix-assisted laser desorption ionization time-of-flight mass spectrometry, circular dichroism, inductively coupled plasma atomic emission spectroscopy, and NMR spectroscopy. We found that the RNA recognition motif (RRM) and zinc finger-like domains exclusively form protease-resistant core structures within the isolated TLS protein fragments, while the remaining regions, including the Arg-Gly-Gly repeats, appear to be completely unstructured. Thus, TLS contains the unstructured N-terminal half followed by the RRM and zinc finger-like domains, which are connected to each other by a flexible linker. We also carried out NMR analyses to obtain more detailed insights into the individual RRM and zinc finger-like domains. The 113Cd NMR analysis of the zinc finger-like domain verified that zinc is coordinated with four cysteines in the C4 type scheme. We also investigated the interaction of each domain with an oligo-RNA containing the GGUG sequence, which appears to be critical for the TLS function in splicing. The backbone amide NMR chemical shift perturbation analyses indicated that the zinc finger domain binds GGUG-containing RNA with a dissociation constant of about 1.0 x 10(-5) m, whereas the RRM domain showed no observable interaction with this RNA. This surprising result implies that the zinc finger domain plays a more predominant role in RNA recognition than the RRM domain.