Dyson Hj

A dynamic knockout reveals that conformational fluctuations influence the chemical step of enzyme catalysis.

An Escherichia coli dihydrofolate reductase mutant is catalytically defective, because motions in the active site are impaired. Conformational dynamics play a key role in enzyme catalysis. Although protein motions have clear implications for ligand flux, a role for dynamics in the chemical step of enzyme catalysis has not been clearly established. We generated a mutant of Escherichia coli dihydrofolate reductase that abrogates millisecond-time-scale fluctuations in the enzyme active site without perturbing its structural and electrostatic preorganization. This dynamic knockout severely impairs hydride transfer. Thus, we have found a link between conformational fluctuations on the millisecond time scale and the chemical step of an enzymatic reaction, with broad implications for our understanding of enzyme mechanisms and for design of novel protein catalysts.

The immunodominant site of a synthetic immunogen has a conformational preference in water for a type-II reverse turn.

Many short synthetic peptides have now been shown to induce antibodies reactive with their cognate sequences in the intact folded protein1–8. Aside from the usefulness of such antibodies as site-specific reagents, the frequency with which this recognition occurs has raised several theoretical issues, the central one being that of how an antibody to a short synthetic peptide, which represents one of the most disordered states of a site in a protein, can react with the more ordered version of the same sequence in the folded protein. This apparent paradox can be resolved if the target site on the protein approaches disorder or if the peptide in solution or on a carrier adopts, with significant frequency, a conformation compatible with that of the cognate site in the protein. Various studies already suggest that antigenic sites in proteins correspond to regions of high atomic mobility1,9–15. We now show, using high-field nuclear magnetic resonance (NMR) spectroscopy, that a nonapeptide selected by several monoclonal antibodies as the immunodominant site of a 36-amino-acid immunogen (residues 75–110 of influenza virus haemagglutinin16,17) adopts a highly populated type-II reverse-turn conformation in water. This suggests that in this case the antibodies have selected a sequence possessing a conformational preference. Apart from helping us to understand immunological recognition, anti-peptide antibodies may provide reagents of sufficient precision for an immunological approach to the problem of protein folding18–23.

Two different neurodegenerative diseases caused by proteins with similar structures

The downstream prion-like protein (doppel, or Dpl) is a paralog of the cellular prion protein, PrPC. The two proteins have ≈25% sequence identity, but seem to have distinct physiologic roles. Unlike PrPC, Dpl does not support prion replication; instead, overexpression of Dpl in the brain seems to cause a completely different neurodegenerative disease. We report the solution structure of a fragment of recombinant mouse Dpl (residues 26–157) containing a globular domain with three helices and a small amount of β-structure. Overall, the topology of Dpl is very similar to that of PrPC. Significant differences include a marked kink in one of the helices in Dpl, and a different orientation of the two short β-strands. Although the two proteins most likely arose through duplication of a single ancestral gene, the relationship is now so distant that only the structures retain similarity; the functions have diversified along with the sequence.

Three-dimensional structure of a type VI turn in a linear peptide in water solution. Evidence for stacking of aromatic rings as a major stabilizing factor.

Structures have been calculated for the folded conformation found at high population in the cis isomeric form of the peptide NH3(+)-Ser-Tyr-Pro-Phe-Asp-Val-COO- (SYPFDV) in aqueous solution, using distance geometry and restrained molecular dynamics. A number of NMR parameters, including NOE distance restraints and phi and chi 1 dihedral angle restraints derived from coupling constants were used in the calculation. The restraints were carefully selected to exclude those that might have a significant contribution from the unfolded states of the peptide, so that the calculated structure represents only the folded form (present at about 70% population) and contains no information on the other members of the conformational ensemble. The calculations give a tight family of structures in the region Tyr2 to Phe4, all containing a type VI turn with an unusual disposition of the aromatic rings of Tyr and Phe, which stack against the proline ring. Both backbone and side-chain conformations are well defined in this region. At the ends of the molecule the polypeptide chain is disordered. The structures are consistent with all of the available NMR information, including upfield chemical shifts observed for the proline ring protons in the cis isomer, and with the independently determined proline ring pucker. There is no evidence for cross-turn hydrogen bonding. According to the calculated structures, the major source of stabilization of the turn conformation appears to be the stacking of the aromatic and proline rings.

Use of chemical shifts and coupling constants in nuclear magnetic resonance structural studies on peptides and proteins.

Publisher Summary This chapter describes advances in the use of coupling constants and chemical shifts in structural studies of peptides and proteins. Coupling constant information can be incorporated into the input data for structure calculations, giving direct information on dihedral angles and stereospecific assignments of prochiral groups such as β-methylene protons and the methyl groups of leucine and valine. Because of the complexity of the interactions that influence the chemical shift of each nucleus, this type of information is more useful at the structure refinement stage. However, the determination of protein and peptide structure using information from nuclear magnetic resonance (NMR) experiments in solution is now a well-established method, and it is frequently used as an adjunct to X-ray crystal structure determination, as well as in cases where crystals are unavailable. With increasing size of the proteins for which three-dimensional (3D) structure determination by NMR is attempted, there will be an increasing reliance on 3D and higher dimensional spectroscopy utilizing transfer pathways selective for particular coupling constants. The method of J doubling is suggested to increase accuracy of coupling constant measurements in large molecules.

Divergent evolution of protein conformational dynamics in dihydrofolate reductase.

Molecular evolution is driven by mutations, which may affect the fitness of an organism and are then subject to natural selection or genetic drift. Analysis of primary protein sequences and tertiary structures has yielded valuable insights into the evolution of protein function, but little is known about the evolution of functional mechanisms, protein dynamics and conformational plasticity essential for activity. We characterized the atomic-level motions across divergent members of the dihydrofolate reductase (DHFR) family. Despite structural similarity, Escherichia coli and human DHFRs use different dynamic mechanisms to perform the same function, and human DHFR cannot complement DHFR-deficient E. coli cells. Identification of the primary-sequence determinants of flexibility in DHFRs from several species allowed us to propose a likely scenario for the evolution of functionally important DHFR dynamics following a pattern of divergent evolution that is tuned by cellular environment.

Role of Intrinsic Protein Disorder in the Function and Interactions of the Transcriptional Coactivators CREB-binding Protein (CBP) and p300

The transcriptional coactivators CREB-binding protein (CBP) and p300 undergo a particularly rich set of interactions with disordered and partly ordered partners, as a part of their ubiquitous role in facilitating transcription of genes. CBP and p300 contain a number of small structured domains that provide scaffolds for the interaction of disordered transactivation domains from a wide variety of partners, including p53, hypoxia-inducible factor 1α (HIF-1α), NF-κB, and STAT proteins, and are the targets for the interactions of disordered viral proteins that compete with cellular factors to disrupt signaling and subvert the cell cycle. The functional diversity of the CBP/p300 interactome provides an excellent example of the power of intrinsic disorder to facilitate the complexity of living systems.

Recognition of the disordered p53 transactivation domain by the transcriptional adapter zinc finger domains of CREB-binding protein

Significance The tumor suppressor p53 regulates the cellular response to genomic damage by recruiting the transcriptional coactivator cyclic-AMP response element-binding protein (CREB)-binding protein (CBP) and its paralog p300 to activate stress response genes. We report NMR structures of the complexes formed between the full-length, intrinsically disordered N-terminal transactivation domain of p53 and the transcriptional adapter zinc finger domains (TAZ1 and TAZ2) of CBP. Exchange broadening of NMR spectra of the complexes was ameliorated by using fusion proteins and segmental isotope labeling. The structures show how the p53 transactivation domain uses bipartite binding motifs to recognize diverse partners, reveal the critical interactions required for high affinity binding, and provide insights into the mechanism by which phosphorylation enhances the ability of p53 to recruit CBP and p300. An important component of the activity of p53 as a tumor suppressor is its interaction with the transcriptional coactivators cyclic-AMP response element-binding protein (CREB)-binding protein (CBP) and p300, which activate transcription of p53-regulated stress response genes and stabilize p53 against ubiquitin-mediated degradation. The highest affinity interactions are between the intrinsically disordered N-terminal transactivation domain (TAD) of p53 and the TAZ1 and TAZ2 domains of CBP/p300. The NMR spectra of simple binary complexes of the TAZ1 and TAZ2 domains with the p53TAD suffer from exchange broadening, but innovations in construct design and isotopic labeling have enabled us to obtain high-resolution structures using fusion proteins, uniformly labeled in the case of the TAZ2–p53TAD fusion and segmentally labeled through transintein splicing for the TAZ1–p53TAD fusion. The p53TAD is bipartite, with two interaction motifs, termed AD1 and AD2, which fold to form short amphipathic helices upon binding to TAZ1 and TAZ2 whereas intervening regions of the p53TAD remain flexible. Both the AD1 and AD2 motifs bind to hydrophobic surfaces of the TAZ domains, with AD2 making more extensive hydrophobic contacts consistent with its greater contribution to the binding affinity. Binding of AD1 and AD2 is synergistic, and structural studies performed with isolated motifs can be misleading. The present structures of the full-length p53TAD complexes demonstrate the versatility of the interactions available to an intrinsically disordered domain containing bipartite interaction motifs and provide valuable insights into the structural basis of the affinity changes that occur upon stress-related posttranslational modification.

Side-chain conformational heterogeneity of intermediates in the Escherichia coli dihydrofolate reductase catalytic cycle.

Escherichia coli dihydrofolate reductase (DHFR) provides a paradigm for the integrated study of the role of protein dynamics in enzyme function. Previous studies of backbone and side chain dynamics have yielded unprecedented insights into the mechanism by which DHFR progresses through the structural changes that occur during its catalytic cycle. Here we report a comprehensive study of the χ1 rotamer populations of the aromatic and γ-methyl containing residues for complexes of the catalytic cycle, based on NMR measurement of (3)JCγCO and (3)JCγN coupling constants. We report conformational and dynamic information for eight distinct complexes, where transitions between rotamer wells may occur on a broad picosecond to millisecond time scale. This large volume of (3)J data has allowed us to fit new Karplus parameterizations for aromatic side chains and to select the best available of previously determined parameters for Ile, Thr, and Val. The (3)JCγCO and (3)JCγN coupling constants are found to be extremely sensitive measures of side chain χ1 rotamers and to give important insights into the extent of conformational averaging. For a subset of residues in DHFR, the extent of rotamer averaging is invariant to the nature of the bound ligand, while for other residues the rotamer averaging differs in one or more complexes of the enzymatic cycle. These variable-averaging residues are generally located near the active site, but the phenomenon extends into the adenosine binding domain. For several residues, the rotamer populations in different DHFR complexes appear to depend on whether the complex is in the closed or occluded state, and some residues are exquisitely sensitive to small changes in the nature of the bound ligand.

Mapping Protein Folding Landscapes by NMR Relaxation

The process of protein folding provides an excellent example of the interactions of water with biomolecules. The changes in the water–protein interactions along the protein folding pathway provide an important impetus for the formation of the final natively folded structure of the protein. NMR spectroscopy provides unique insights into the dynamic protein folding process, and during the past 20 years we have seen the development of a wide range of NMR techniques to probe the kinetic and thermodynamic aspects of protein folding. In particular, with the advent of high-field spectrometers and stable isotope labeling techniques, the structure and dynamics of a wide range of disordered and partly ordered proteins at equilibrium have been characterized by NMR. Efforts in our laboratory over a number of years have allowed the sequence-specific identification of sites of local hydrophobic collapse, as well as secondary structure formation and transient long-range interactions in several protein systems, most notably for apomyoglobin, which will be highlighted in this article.