Tandem affinity purification combined with inducible shRNA expression as a tool to study the maturation of macromolecular assemblies

Tandem affinity purification (TAP) is an efficient method for the purification and characterization of large macromolecular complexes. To elucidate the role of specific components of such complexes, it is important to address the question of how loss of a specific factor affects complex composition. Here, we introduce a method that combines TAP of large macromolecular assemblies with inducible shRNA-mediated protein depletion in human somatic cells. As a proof of principle, we have applied this method to the purification of human pre-ribosomal particles. Using inducible expression of ribosome assembly factors as bait proteins, different pre-40S particles could be isolated and characterized, revealing high conservation of the ribosome biogenesis pathway from yeast to human cells. Besides known ribosome maturation factors, C21orf70 was identified as a novel pre-40S component. By combining TAP of pre-40S particles with shRNA-mediated depletion of the pre-40S-associated protein kinase Rio2, we observed that increased levels of the nuclear HEAT-repeat protein Rrp12 are associated with 40S precursors in absence of Rio2. Further analyses revealed that Rrp12 is partially mislocalized to the cytoplasm and trapped on late 40S precursors upon loss of Rio2, and therefore fails to efficiently recycle to the nucleus. Thus, the combination of tandem affinity purification and shRNA induction provided further insights into late cytoplasmic 40S maturation steps, demonstrating the high potential of this method.     

The Syk-binding ubiquitin ligase c-Cbl mediates signaling-dependent B cell receptor ubiquitination and B cell receptor-mediated antigen processing and presentation

B cell receptor (BCR)-mediated antigen (Ag) processing and presentation lead to B cell-T cell interactions, which support affinity maturation and immunoglobulin class switching. These interactions are supported by generation of peptide-MHC class II complexes in multivesicular body-like MIIC compartments of B cells. Previous studies have shown that trafficking of Ag·BCR complexes to MVB-like MIIC occurs via an ubiquitin-dependent pathway and that ubiquitination of Ag·BCR complexes occurs by an Src family kinase signaling-dependent mechanism that is restricted to lipid raft-resident Ag·BCR complexes. This study establishes that downstream Syk-dependent BCR signaling is also required for BCR ubiquitination and BCR-mediated antigen processing and presentation. Knockdown studies reveal that of the two known Syk-binding E3 ubiquitin ligases c-Cbl and Cbl-b, only c-Cbl appears to have a central role in BCR ubiquitination, trafficking to MIIC, and ubiquitin-dependent BCR-mediated antigen processing and presentation. These results establish the novel role for Syk signaling and the Syk-binding ubiquitin ligase c-Cbl in the BCR-mediated processing and presentation of cognate antigen and define one mechanism by which antigen-induced BCR ubiquitination is modulated to impact the initiation and maturation of the humoral immune response.     

The Structural Pathway of Interleukin 1 (IL-1) Initiated Signaling Reveals Mechanisms of Oncogenic Mutations and SNPs in Inflammation and Cancer

Interleukin-1 (IL-1) is a large cytokine family closely related to innate immunity and inflammation. IL-1 proteins are key players in signaling pathways such as apoptosis, TLR, MAPK, NLR and NF-κB. The IL-1 pathway is also associated with cancer, and chronic inflammation increases the risk of tumor development via oncogenic mutations. Here we illustrate that the structures of interfaces between proteins in this pathway bearing the mutations may reveal how. Proteins are frequently regulated via their interactions, which can turn them ON or OFF. We show that oncogenic mutations are significantly at or adjoining interface regions, and can abolish (or enhance) the protein-protein interaction, making the protein constitutively active (or inactive, if it is a repressor). We combine known structures of protein-protein complexes and those that we have predicted for the IL-1 pathway, and integrate them with literature information. In the reconstructed pathway there are 104 interactions between proteins whose three dimensional structures are experimentally identified; only 15 have experimentally-determined structures of the interacting complexes. By predicting the protein-protein complexes throughout the pathway via the PRISM algorithm, the structural coverage increases from 15% to 71%. In silico mutagenesis and comparison of the predicted binding energies reveal the mechanisms of how oncogenic and single nucleotide polymorphism (SNP) mutations can abrogate the interactions or increase the binding affinity of the mutant to the native partner. Computational mapping of mutations on the interface of the predicted complexes may constitute a powerful strategy to explain the mechanisms of activation/inhibition. It can also help explain how an oncogenic mutation or SNP works.     

Structural basis of affinity maturation and intramolecular cooperativity in a protein-protein interaction

Although protein-protein interactions are involved in nearly all cellular processes, general rules for describing affinity and selectivity in protein-protein complexes are lacking, primarily because correlations between changes in protein structure and binding energetics have not been well determined. Here, we establish the structural basis of affinity maturation for a protein-protein interaction system that we had previously characterized energetically. This model system exhibits a 1500-fold affinity increase. Also, its affinity maturation is restricted by negative intramolecular cooperativity. With three complex and six unliganded variant X-ray crystal structures, we provide molecular snapshots of protein interface remodeling events that span the breadth of the affinity maturation process and present a comprehensive structural view of affinity maturation. Correlating crystallographically observed structural changes with measured energetic changes reveals molecular bases for affinity maturation, intramolecular cooperativity, and context-dependent binding.     

Specific Fluorine Labeling of the HyHEL10 Antibody Affects Antigen Binding and Dynamics

To more fully understand the molecular mechanisms responsible for variations in binding affinity with antibody maturation, we explored the use of site specific fluorine labeling and (19)F nuclear magnetic resonance (NMR). Several single-chain (scFv) antibodies, derived from an affinity-matured series of anti-hen egg white lysozyme (HEL) mouse IgG1, were constructed with either complete or individual replacement of tryptophan residues with 5-fluorotryptophan ((5F)W). An array of biophysical techniques was used to gain insight into the impact of fluorine substitution on the overall protein structure and antigen binding. SPR measurements indicated that (5F)W incorporation lowered binding affinity for the HEL antigen. The degree of analogue impact was residue-dependent, and the greatest decrease in affinity was observed when (5F)W was substituted for residues near the binding interface. In contrast, corresponding crystal structures in complex with HEL were essentially indistinguishable from the unsubstituted antibody. (19)F NMR analysis showed severe overlap of signals in the free fluorinated protein that was resolved upon binding to antigen, suggesting very distinct chemical environments for each (5F)W in the complex. Preliminary relaxation analysis suggested the presence of chemical exchange in the antibody-antigen complex that could not be observed by X-ray crystallography. These data demonstrate that fluorine NMR can be an extremely useful tool for discerning structural changes in scFv antibody-antigen complexes with altered function that may not be discernible by other biophysical techniques.     

Sequential protein association with nascent 60S ribosomal particles

Ribosome biogenesis in eukaryotes depends on the coordinated action of ribosomal and nonribosomal proteins that guide the assembly of preribosomal particles. These intermediate particles follow a maturation pathway in which important changes in their protein composition occur. The mechanisms involved in the coordinated assembly of the ribosomal particles are poorly understood. We show here that the association of preribosomal factors with pre-60S complexes depends on the presence of earlier factors, a phenomenon essential for ribosome biogenesis. The analysis of the composition of purified preribosomal complexes blocked in maturation at specific steps allowed us to propose a model of sequential protein association with, and dissociation from, early pre-60S complexes for several preribosomal factors such as Mak11, Ssf1, Rlp24, Nog1, and Nog2. The presence of either Ssf1 or Nog2 in complexes that contain the 27SB pre-rRNA defines novel, distinct pre-60S particles that contain the same pre-rRNA intermediates and that differ only by the presence or absence of specific proteins. Physical and functional interactions between Rlp24 and Nog1 revealed that the assembly steps are, at least in part, mediated by direct protein-protein interactions.     

X-ray snapshots of the maturation of an antibody response to a protein antigen

The process whereby the immune system generates antibodies of higher affinities during a response to antigen (affinity maturation) is a prototypical example of molecular evolution. Earlier studies have been confined to antibodies specific for small molecules (haptens) rather than for proteins. We compare the structures of four antibodies bound to the same site on hen egg white lysozyme (HEL) at different stages of affinity maturation. These X-ray snapshots reveal that binding is enhanced, not through the formation of additional hydrogen bonds or van der Waals contacts or by an increase in total buried surface, but by burial of increasing amounts of apolar surface at the expense of polar surface, accompanied by improved shape complementarity. The increase in hydrophobic interactions results from highly correlated rearrangements in antibody residues at the interface periphery, adjacent to the central energetic hot spot. This first visualization of the maturation of antibodies to protein provides insights into the evolution of high affinity in other protein-protein interfaces.     

Structural basis of HIV-1 neutralization by affinity matured Fabs directed against the internal trimeric coiled-coil of gp41

The conserved internal trimeric coiled-coil of the N-heptad repeat (N-HR) of HIV-1 gp41 is transiently exposed during the fusion process by forming a pre-hairpin intermediate, thus representing an attractive target for the design of fusion inhibitors and neutralizing antibodies. In previous studies we reported a series of broadly neutralizing mini-antibodies derived from a synthetic naïve human combinatorial antibody library by panning against a mimetic of the trimeric N-HR coiled coil, followed by affinity maturation using targeted diversification of the CDR-H2 loop. Here we report crystal structures of the N-HR mimetic 5-Helix with two Fabs that represent the extremes of this series: Fab 8066 is broadly neutralizing across a wide panel of B and C type HIV-1 viruses, whereas Fab 8062 is non-neutralizing. The crystal structures reveal important differences in the conformations of the CDR-H2 loops in the complexes that propagate into other regions of the antigen-antibody interface, and suggest that both neutralization properties and affinity for the target can be attributed, at least in part, to the differences in the interactions of the CDR-H2 loops with the antigen. Furthermore, modeling of the complex of an N-HR trimer with three Fabs suggests that the CDR-H2 loop may be involved in close intermolecular contacts between neighboring antibody molecules, and that such contacts may hinder the formation of complexes between the N-HR trimer and more than one antibody molecule depending on the conformation of the bound CDR-H2 loop which is defined by its interactions with antigen. Comparison with the crystal structure of the complex of 5-Helix with another neutralizing monoclonal antibody known as D5, derived using an entirely different antibody library and panning procedure, reveals remarkable convergence in the optimal sequence and conformation of the CDR-H2 loop.     

WT p53, but not tumor-derived mutants, bind to Bcl2 via the DNA binding domain and induce mitochondrial permeabilization

The induction of apoptosis by p53 in response to cellular stress is its most conserved function and crucial for p53 tumor suppression. We recently reported that p53 directly induces oligomerization of the BH1,2,3 effector protein Bak, leading to outer mitochondrial membrane permeabilization (OMMP) with release of apoptotic activator proteins. One important mechanism by which p53 achieves OMMP is by forming an inhibitory complex with the anti-apoptotic BclXL protein. In contrast, the p53 complex with the Bcl2 homolog has not been interrogated. Here we have undertaken a detailed characterization of the p53-Bcl2 interaction using structural, biophysical, and mutational analyses. We have identified the p53 DNA binding domain as the binding interface for Bcl2 using solution NMR. The affinity of the p53-Bcl2 complex was determined by surface plasmon resonance analysis (BIAcore) to have a dominant component KD 535 +/- 24 nm. Moreover, in contrast to wild type p53, endogenous missense mutants of p53 are unable to form complexes with endogenous Bcl2 in human cancer cells. Functionally, these mutants are all completely or strongly compromised in mediating OMMP, as measured by cytochrome c release from isolated mitochondria. These data implicate p53-Bcl2 complexes in contributing to the direct mitochondrial p53 pathway of apoptosis and further support the notion that the DNA binding domain of p53 is a dual function domain, mediating both its transactivation function and its direct mitochondrial apoptotic function.     

Changes in the stability and biomechanics of P22 bacteriophage capsid during maturation

The capsid of P22 bacteriophage undergoes a series of structural transitions during maturation that guide it from spherical to icosahedral morphology. The transitions include the release of scaffold proteins and capsid expansion. Although P22 maturation has been investigated for decades, a unified model that incorporates thermodynamic and biophysical analyses is not available. A general and specific model of icosahedral capsid maturation is of significant interest to theoreticians searching for fundamental principles as well as virologists and material scientists seeking to alter maturation to their advantage. To address this challenge, we have combined the results from orthogonal biophysical techniques including differential scanning fluorimetry, atomic force microscopy, circular dichroism, and hydrogen-deuterium exchange mass spectrometry. By integrating these results from single particle and population measurements, an energy landscape of P22 maturation from procapsid through expanded shell to wiffle ball emerged, highlighting the role of metastable structures and the thermodynamics guiding maturation. The propagation of weak quaternary interactions across symmetric elements of the capsid is a key component for stability in P22. A surprising finding is that the progression to wiffle ball, which lacks pentamers, shows that chemical and thermal stability can be uncoupled from mechanical rigidity, elegantly demonstrating the complexity inherent in capsid protein interactions and the emergent properties that can arise from icosahedral symmetry. On a broader scale, this work demonstrates the power of applying orthogonal biophysical techniques to elucidate assembly mechanisms for supramolecular complexes and provides a framework within which other viral systems can be compared.     

A structure-based benchmark for protein-protein binding affinity

We have assembled a nonredundant set of 144 protein-protein complexes that have high-resolution structures available for both the complexes and their unbound components, and for which dissociation constants have been measured by biophysical methods. The set is diverse in terms of the biological functions it represents, with complexes that involve G-proteins and receptor extracellular domains, as well as antigen/antibody, enzyme/inhibitor, and enzyme/substrate complexes. It is also diverse in terms of the partners' affinity for each other, with K(d) ranging between 10(-5) and 10(-14) M. Nine pairs of entries represent closely related complexes that have a similar structure, but a very different affinity, each pair comprising a cognate and a noncognate assembly. The unbound structures of the component proteins being available, conformation changes can be assessed. They are significant in most of the complexes, and large movements or disorder-to-order transitions are frequently observed. The set may be used to benchmark biophysical models aiming to relate affinity to structure in protein-protein interactions, taking into account the reactants and the conformation changes that accompany the association reaction, instead of just the final product.     

The influence of immune complex-bearing follicular dendritic cells on the IgM response, Ig class switching, and production of high affinity IgG

It is believed that Ag in immune complexes (ICs) on follicular dendritic cells (FDCs) selects high affinity B cells and promotes affinity maturation. However, selection has been documented in the absence of readily detectable ICs on FDCs, suggesting that FDC-ICs may not be important. These results prompted experiments to test the hypothesis that IC-bearing murine FDCs can promote high affinity IgG responses by selecting B cells after stimulating naive IgM(+) cells to mature and class switch. Coculturing naive lambda(+) B cells, FDCs, (4-hydroxy-3-nitrophenyl)acetyl-chicken gamma-globulin (CGG) + anti-CGG ICs, and CGG-primed T cells resulted in FDC-lymphocyte clusters and production of anti-4-hydroxy-5-iodo-3-nitrophenyl acetyl. Class switching was indicated by a shift from IgM to IgG, and affinity maturation was indicated by a change from mostly low affinity IgM and IgG in the first week to virtually all high affinity IgG anti-4-hydroxy-5-iodo-3-nitrophenyl acetyl in the second week. Class switching and affinity maturation were easily detectable in the presence of FDCs bearing appropriate ICs, but not in the absence of FDCs. Free Ag plus FDCs resulted in low affinity IgG, but affinity maturation was only apparent when FDCs bore ICs. Class switching is activation-induced cytidine deaminase (AID) dependent, and blocking FDC-CD21 ligand-B cell CD21 interactions inhibited FDC-IC-mediated enhancement of AID production and the IgG response. In short, these data support the concept that ICs on FDCs can promote AID production, class switching, and maturation of naive IgM(+) B cells, and further suggest that the IC-bearing FDCs help select high affinity B cells that produce high affinity IgG.     

Reassessing the function of immune-complex retention by follicular dendritic cells

The close association of follicular dendritic cells (FDCs) and germinal-centre B cells has fostered the idea that B-cell recognition of retained antigen that is presented on the surface of FDCs is important for affinity maturation and memory B-cell development. We argue that the retention of immune complexes is not required for germinal-centre development, affinity maturation and memory B-cell maintenance. Instead, it is probable that FDCs support B-cell proliferation and differentiation in a non-specific manner. Other potential roles of immune complexes retained by FDCs are discussed.     

Nog2p, a putative GTPase associated with pre-60S subunits and required for late 60S maturation steps.

Eukaryotic ribosome maturation depends on a set of well ordered processing steps. Here we describe the functional characterization of yeast Nog2p (Ynr053cp), a highly conserved nuclear protein. Nog2p contains a putative GTP-binding site, which is essential in vivo. Kinetic and steady-state measurements of the levels of pre-rRNAs in Nog2p-depleted cells showed a defect in 5.8S and 25S maturation and a concomitant increase in the levels of both 27SB(S) and 7S(S) precursors. We found Nog2p physically associated with large pre-60S complexes highly enriched in the 27SB and 7S rRNA precursors. These complexes contained, besides a subset of ribosomal proteins, at least two additional factors, Nog1p, another putative GTP-binding protein, and Rlp24p (Ylr009wp), which belongs to the Rpl24e family of archaeal and eukaryotic ribosomal proteins. In the absence of Nog2p, the pre-60S ribosomal complexes left the nucleolus, but were retained in the nucleoplasm. These results suggest that transient, possibly GTP-dependent association of Nog2p with the pre-ribosomes might trigger late rRNA maturation steps in ribosomal large subunit biogenesis.     

Dissecting cooperative and additive binding energetics in the affinity maturation pathway of a protein-protein interface

When two proteins associate they form a molecular interface that is a structural and energetic mosaic. Within such interfaces, individual amino acid residues contribute distinct binding energies to the complex. In combination, these energies are not necessarily additive, and significant positive or negative cooperative effects often exist. The basis of reliable algorithms to predict the specificities and energies of protein-protein interactions depends critically on a quantitative understanding of this cooperativity. We have used a model protein-protein system defined by an affinity maturation pathway, comprising variants of a T cell receptor Vbeta domain that exhibit an overall affinity range of approximately 1500-fold for binding to the superantigen staphylococcal enterotoxin C3, in order to dissect the cooperative and additive energetic contributions of residues within an interface. This molecular interaction has been well characterized previously both structurally, by x-ray crystallographic analysis, and energetically, by scanning alanine mutagenesis. Through analysis of group and individual maturation and reversion mutations using surface plasmon resonance spectroscopy, we have identified energetically important interfacial residues, determined their cooperative and additive energetic properties, and elucidated the kinetic and thermodynamic bases for molecular evolution in this system. The summation of the binding free energy changes associated with the individual mutations that define this affinity maturation pathway is greater than that of the fully matured variant, even though the affinity gap between the end point variants is relatively large. Two mutations in particular, both located in the complementarity determining region 2 loop of the Vbeta domain, exhibit negative cooperativity.     

Expression, purification and characterization of the recombinant ribonuclease P protein component from Bacillus subtilis.

Ribonuclease P is a ribonucleoprotein complex that catalyzes the essential 5' maturation of all precursor tRNA molecules. The protein component both alters the conformation of the RNA component and enhances the substrate affinity and specificity. To facilitate biochemical and biophysical studies, the protein component of Bacillus subtilis ribonuclease P (RNase P) was overproduced in Escherichia coli using the native amino acid sequence with the initial 20 codons optimized for expression in E.coli . A simple purification procedure using consecutive cation exchange chromatography steps in the presence and absence of urea was developed to purify large quantities of P protein without contaminating nucleic acids. The identity of the recombinant protein as a cofactor of RNase P was established by its ability to stimulate the activity of the RNA component in low ionic strength buffer in a 1:1 stoichiometry. Circular dichroism studies indicate that P protein is a combination of alpha-helix and beta-sheet secondary structures and is quite stable, with a T m of 67 degrees C. The described methods facilitated the large scale purification of homogeneous, RNA-free P protein required for high resolution crystallographic analyses and may be useful for the preparation of other RNA binding proteins.