Mapping the ligand binding pocket in the cellular retinaldehyde binding protein

Retinoid interactions determine the function of the cellular retinaldehyde binding protein (CRALBP) in the rod visual cycle where it serves as an 11-cis-retinol acceptor for the enzymatic isomerization of all-trans- to 11-cis-retinol and as a substrate carrier for 11-cis-retinol dehydrogenase (RDH5). Based on preliminary NMR studies suggesting retinoid interactions with Met and Trp residues, human recombinant CRALBP (rCRALBP) with altered Met or Trp were produced and analyzed for ligand interactions. The primary structures of the purified proteins were verified for mutants M208A, M222A, M225A, W165F, and W244F, then retinoid binding properties and substrate carrier functions were evaluated. All the mutant proteins bound 11-cis- and 9-cis-retinal and therefore were not grossly misfolded. Altered UV-visible spectra and lower retinoid binding affinities were observed for the mutants, supporting modified ligand interactions. Altered kinetic parameters were observed for RDH5 oxidation of 11-cis-retinol bound to rCRALBP mutants M222A, M225A, and W244F, supporting impaired substrate carrier function. Heteronuclear single quantum correlation NMR analyses confirmed localized structural changes upon photoisomerization of rCRALBP-bound 11-cis-retinal and demonstrated ligand-dependent conformational changes for residues Met-208, Met-222, Trp-165, and Trp-244. Furthermore, residues Met-208, Met-222, Met-225, and Trp-244 are within a region exhibiting high homology to the ligand binding cavity of phosphatidylinositol transfer protein. Overall the data implicate Trp-165, Met-208, Met-222, Met-225, and Trp-244 as components of the CRALBP ligand binding cavity.     

Disease-causing SAP mutants are defective in ligand binding and protein folding

The X-linked lymphoproliferative (XLP) syndrome is caused by mutations or deletions in the SH2D1A gene that encodes an SH2 domain protein named SH2D1A or SAP. The identification of a number of missense mutations within the protein's SH2 domain, each of which can directly cause disease, provides a unique opportunity to investigate the function of an interaction protein module, SH2, in the pathogenesis of XLP. We show here that SAP mutants found in XLP patients are defective in binding its physiological ligands signaling lymphocyte activating molecule (SLAM), a co-receptor in T cell activation, and Fyn, a Src family protein tyrosine kinase. Consequently, these mutants are deficient in signaling through the SLAM receptor. This is reflected by compromised abilities for the mutants to recruit Fyn to SLAM and to activate Fyn, by reduced phosphorylation of the receptor, and by deficiencies for the mutants in blocking binding of SHP-2 to SLAM. Furthermore, all mutants examined are defective in protein folding as manifested by their significantly reduced melting temperatures upon thermal denaturation, compared to that of SAP. Taken together, these results suggest that defects in ligand binding, receptor signaling, and protein folding collectively contribute to the loss of function for disease-causing SAP mutants.     

Disease-causing mutations in cartilage oligomeric matrix protein cause an unstructured Ca2+ binding domain.

Chondrocytes from pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (EDM1) patients display an enlarged rough endoplasmic reticulum that accumulates extracellular matrix proteins, including cartilage oligomeric matrix protein (COMP). Mutations that cause PSACH and EDM1 are restricted to a 27-kDa Ca(2+) binding domain (type 3 repeat). This domain has 13 Ca(2+)-binding loops with a consensus sequence that conforms to Ca(2+)-binding loops found in EF hands. Most disease-causing mutations are found in the 11-kDa C-terminal region of this domain. We expressed recombinant native and mutant forms of the type 3 repeat domain (T3) and its 11-kDa C-terminal region (T3-Cterm). T3 and T3-Cterm bind approximately 13 and 8 mol of Ca(2+)/mol of protein, respectively. CD, one-dimensional proton, and two-dimensional (1)H-(15)N HSQC spectra of Ca(2+)-bound T3-Cterm indicate a distinct conformation that has little helical secondary structure, despite the presence of 13 EF hand Ca(2+)-binding loops. This conformation is also formed within the context of the intact T3. 19 cross-peaks found between 9.0 and 11.4 ppm are consistent with the presence of strong hydrogen bonding patterns, such as those in beta-sheets. Removal of Ca(2+) leads to an apparent loss of structure as evidenced by decreased dispersion and loss of all down field resonances. Deletion of Asp-470 (a mutation found in 22% of all PSACH and EDM1 patients) decreased the Ca(2+)-binding capacity of both T3 and T3-Cterm by about 3 mol of Ca(2+)/mol of protein. Two-dimensional (1)H-(15)N HSQC spectra of mutated T3-Cterm showed little evidence of defined structure in the presence or absence of Ca(2+). The data demonstrate that Ca(2+) is required to nucleate folding and to maintain defined structure. Mutation results in a partial loss of Ca(2+)-binding capacity and prevents Ca(2+)-dependent folding. Persistence of an unstructured state of the mutated Ca(2+) binding domain in COMP is the structural basis for retention of COMP in the rough endoplasmic reticulum of differentiated PSACH and EDM1 chondrocytes.     

Visual cycle impairment in cellular retinaldehyde binding protein (CRALBP) knockout mice results in delayed dark adaptation

Mutations in the human CRALBP gene cause retinal pathology and delayed dark adaptation. Biochemical studies have not identified the primary physiological function of CRALBP. To resolve this, we generated and characterized mice with a non-functional CRALBP gene (Rlbp1(-/-) mice). The photosensitivity of Rlbp1(-/-) mice is normal but rhodopsin regeneration, 11-cis-retinal production, and dark adaptation after illumination are delayed by >10-fold. All-trans-retinyl esters accumulate during the delay indicating that isomerization of all-trans- to 11-cis-retinol is impaired. No evidence of photoreceptor degeneration was observed in animals raised in cyclic light/dark conditions for up to 1 year. Albino Rlbp(-/-) mice are protected from light damage relative to the wild type. These findings support a role for CRALBP as an acceptor of 11-cis-retinol in the isomerization reaction of the visual cycle.     

Disease-causing mutations in genes of the complement system

Recent studies have revealed profound developmental consequences of mutations in genes encoding proteins of the lectin pathway of complement activation, a central component of the innate immune system. Apart from impairment of immunity against microorganisms, it is known that hereditary deficiencies of this system predispose one to autoimmune conditions. Polymorphisms in complement genes are linked to, for example, atypical hemolytic uremia and age-dependent macular degeneration. The complement system comprises three convergent pathways of activation: the classical, the alternative, and the lectin pathway. The recently discovered lectin pathway is less studied, but polymorphisms in the plasma pattern-recognition molecule mannan-binding lectin (MBL) are known to impact its level, and polymorphisms in the MBL-associated serine protease-2 (MASP-2) result in defects of complement activation. Recent studies have described roles outside complement and immunity of another MBL-associated serine protease, MASP-3, in the etiology of 3MC syndrome, an autosomal-recessive disorder involving a spectrum of developmental features, including characteristic facial dysmorphism. Syndrome-causing mutations were identified in MASP1, encoding MASP-3 and two additional proteins, MASP-1 and MAp44. Furthermore, an association was discovered between 3MC syndrome and mutations in COLEC11, encoding CL-K1, another molecule of the lectin pathway. The findings were confirmed in zebrafish, indicating that MASP-3 and CL-K1 underlie an evolutionarily conserved pathway of embryonic development. Along with the discovery of a role of C1q in pruning synapses in mice, these recent advances point toward a broader role of complement in development. Here, we compare the functional immunologic consequences of “conventional” complement deficiencies with these newly described developmental roles.     

Effects of ligand binding on dynamics of fatty acid binding protein and interactions with membranes

Intracellular transport of fatty acids involves binding of ligands to their carrier fatty acid binding proteins (FABPs) and interactions of ligand-free and -bound FABPs with membranes. Previous studies focused on ligand-free FABPs. Here, our amide hydrogen exchange data showed that oleic acid binding to human intestinal FABP (hIFABP) stabilizes the protein, most likely through enhancing the hydrogen-bonding network, and induces rearrangement of sidechains even far away from the ligand binding site. Using NMR relaxation techniques, we found that the ligand binding affects not only conformational exchanges between major and minor states but also the affinity of hIFABP to nanodiscs. Analyses of the relaxation and amide exchange data suggested that two minor native-like states existing in both ligand-free and -bound hIFABPs originate from global “breathing” motions, while one minor native-like state comes from local motions. The amide hydrogen exchange data also indicated that helix αII undergoes local unfolding through which ligands can exit from the binding cavity.     

PII T-loop mutations affecting signal transduction to NtrB also abolish yeast two-hybrid interactions

Mutations A49P and Delta47-53 at the T loop of the Escherichia coli GlnB (PII) protein impair regulatory interactions with the two-component sensor regulator NtrB (P. Jiang, P. Zucker, M. R. Atkinson, E. S. Kamberov, W. Tirasophon, P. Chandran, B. R. Schepke, and A. J. Ninfa, J. Bacteriol. 179: 4342-4353, 1997). We show here that these mutations also impair interactions between PII and NtrB in the yeast two-hybrid system, indicating that defects in NtrB regulation closely reflect binding impairment. The reported results underline the strength of two-hybrid assays for analysis of interactions involving the T loop of PII proteins.     

LIG1 syndrome mutations remodel a cooperative network of ligand binding interactions to compromise ligation efficiency

Human DNA ligase I (LIG1) is the main replicative ligase and it also seals DNA breaks to complete DNA repair and recombination pathways. Immune compromised patients harbor hypomorphic LIG1 alleles encoding substitutions of conserved arginine residues, R771W and R641L, that compromise LIG1 activity through poorly defined mechanisms. To understand the molecular basis of LIG1 syndrome mutations, we determined high resolution X-ray structures and performed systematic biochemical characterization of LIG1 mutants using steady-state and pre-steady state kinetic approaches. Our results unveil a cooperative network of plastic DNA-LIG1 interactions that connect DNA substrate engagement with productive binding of Mg²⁺ cofactors for catalysis. LIG1 syndrome mutations destabilize this network, compromising Mg²⁺ binding affinity, decreasing ligation efficiency, and leading to elevated abortive ligation that may underlie the disease pathology. These findings provide novel insights into the fundamental mechanism by which DNA ligases engage with a nicked DNA substrate, and they suggest that disease pathology of LIG1 syndrome could be modulated by Mg²⁺ levels.     

Dynamics of cellular retinoic acid binding protein I on multiple time scales with implications for ligand binding

Cellular retinoic acid binding protein I (CRABPI) belongs to the family of intracellular lipid binding proteins (iLBPs), all of which bind a hydrophobic ligand within an internal cavity. The structures of several iLBPs reveal minimal structural differences between the apo (ligand-free) and holo (ligand-bound) forms, suggesting that dynamics must play an important role in the ligand recognition and binding processes. Here, a variety of nuclear magnetic resonance (NMR) spectroscopy methods were used to systematically study the dynamics of both apo and holo CRABPI at various time scales. Translational and rotational diffusion constant measurements were used to study the overall motions of the proteins. Both apo and holo forms of CRABPI tend to self-associate at high (1.2 mM) concentrations, while at low concentrations (0.2 mM), they are predominantly monomeric. Rapid amide exchange rate and laboratory frame relaxation rate measurements at two spectrometer field strengths (500 and 600 MHz) were used to probe the internal motions of the individual residues. Several residues in the apo form, notably within the ligand recognition region, exhibit millisecond time scale motions that are significantly arrested in the holo form. In contrast, no significant differences in the high-frequency motions were observed between the two forms. These results provide direct experimental evidence for dynamics-induced ligand recognition and binding at a specifically defined time scale. They also exemplify the importance of dynamics in providing a more comprehensive understanding of how a protein functions.     

Rapid mapping of protein structure,interactions, and ligand binding by misincorporation proton-alkyl exchange

Understanding protein conformation, interactions, and ligand binding is essential to all biological inquiry. We report a novel biochemical technique, called misincorporation proton-alkyl exchange (MPAX), that can be used to footprint protein structure at single amino acid resolution. MPAX exploits translational misincorporation of cysteine residues to generate probes for physical analysis. We apply MPAX to the triosephosphate isomerase (beta/alpha)(8) barrel, accurately determining its substrate-binding site, a protein-protein interaction surface, the solvent-accessible protein surface, and the stability of the barrel. Because MPAX requires only microgram quantities of material and is not limited by protein size, it is ideally suited for proteins not amenable to conventional structural methods, such as membrane proteins, partially folded or insoluble proteins, and large protein complexes.     

Allosteric tertiary interactions preorganize the c-di-GMP riboswitch and accelerate ligand binding

Cyclic diguanylate (c-di-GMP) is a bacterial second messenger important for physiologic adaptation and virulence. Class-I c-di-GMP riboswitches are phylogenetically widespread and thought to mediate pleiotropic genetic responses to the second messenger. Previous studies suggest that the RNA aptamer domain switches from an extended free state to a compact, c-di-GMP-bound conformation in which two helical stacks dock side-by-side. Single molecule fluorescence resonance energy transfer (smFRET) experiments now reveal that the free RNA exists in four distinct populations that differ in dynamics in the extended and docked conformations. In the presence of c-di-GMP and Mg(2+), a stably docked population (>30 min) becomes predominant. smFRET mutant analysis demonstrates that tertiary interactions distal to the c-di-GMP binding site strongly modulate the RNA population structure, even in the absence of c-di-GMP. These allosteric interactions accelerate ligand recognition by preorganizing the RNA, favoring rapid c-di-GMP binding.     

Mutations in the Trp-Ser-X-Trp-Ser motif of the erythropoietin receptor abolish processing, ligand binding, and activation of the receptor.

The erythropoietin receptor (EPOR) is a member of the newly identified cytokine receptor superfamily. A common sequence motif, Trp-Ser-X-Trp-Ser (WSXWS), near the transmembrane domain is highly conserved in this family. To determine the function of this motif, we constructed deletion and insertion mutations in this part of the EPOR and introduced them into an interleukin-3 (IL-3)-dependent hematopoietic Ba/F3 cell line. Cells expressing the wild-type EPOR displayed 1,500 erythropoietin (EPO)-binding sites/cell with a single affinity of about 300 pM and proliferate in the presence of IL-3 or EPO. Ba/F3 cells expressing receptors mutated in the WSXWS motif displayed little EPO binding on the cell surface and did not grow in the presence of EPO. The mutant receptors were retained in the endoplasmic reticulum (ER) and, as such, were unable to bind EPO. A single Gly insertion between the two WS sequences caused defects in receptor structure and function similar to mutations lacking all or part of the WSXWS motif. The EPOR can be activated, resulting in proliferation independent of EPO either by an Arg129 to Cys point mutation or by association with the Friend spleen focus-forming virus (SFFV) envelope glycoprotein gp55. Introduction of the point mutation (Arg129 to Cys) did not activate any of the receptors mutated in the WSXWS motif. Moreover, gp55 did not activate the mutant receptors in Ba/F3 cells. Our study indicates that the WSXWS motif is critical for protein folding, ligand-binding, and signal transduction.     

Transporter-to-trap conversion: a disulfide bond formation in cellular retinoic acid binding protein I mutant triggered by retinoic acid binding irreversibly locks the ligand inside the protein

Transport proteins must bind their ligands reversibly to enable release at the point of delivery, while irreversible binding is usually associated with the extreme cases of ligand sequestration. Protein conformational dynamics is an important modulator of binding kinetics, as increased flexibility in the regions adjacent to the binding site may facilitate both association and dissociation processes. Ligand entry to, and exit from, the internal binding site of the cellular retinoic acid binding protein I (CRABP I) occurs via a flexible portal region, which functions as a dynamic aperture. We designed and expressed a CRABP I mutant (A35C/T57C), in which a small-scale conformational switch caused by the ligand binding event triggers formation of a disulfide bond in the portal region, thereby arresting structural fluctuations and effectively locking the ligand inside the binding cavity. At the same time, no formation of the disulfide bond is observed in the apo form of the mutant, and most characteristics of the mutant, including protein stability, are very similar to those of the wild-type protein in the absence of retinoic acid. The mutation does not alter the kinetics of retinoic acid binding to the protein, although the disulfide formation makes the binding effectively irreversible, as suggested by the absence of retinoic acid transfer from the holo form of the mutant to lipid vesicles in the absence of a reducing agent. Taken together, these data suggest that the disulfide bond formation in the portal region arrests large-scale structural fluctuations, which are required for retinoic acid release from the protein. The unique properties of the CRABP I mutant described in this work can be used to inspire and guide a design of nanodevices for multiple tasks ranging from sequestering small-molecule toxins in both tissue and circulation to nutrient deprivation of pathogens.     

S-Nitrosylation signaling regulates cellular protein interactions

BACKGROUND: S-Nitrosothiols are made by nitric oxide synthases and other metalloproteins. Unlike nitric oxide, S-nitrosothiols are involved in localized, covalent signaling reactions in specific cellular compartments. These reactions are enzymatically regulated. SCOPE: S-Nitrosylation affects interactions involved in virtually every aspect of normal cell biology. This article is part of a Special Issue entitled Regulation of Cellular Processes by S-nitrosylation. MAJOR CONCLUSIONS AND SIGNIFICANCE: S-Nitrosylation is a regulated signaling reaction.     

Stabilization of the FK506 binding protein by ligand binding.

Although the rotamase activity of the FK506 binding protein is inhibited by ligand binding, it is hypothesized that the ligand/protein complex itself may be responsible for the immunosuppressive effects of FK506. We have therefore examined the structure of the FK506 binding protein in the presence of an analog of FK506 (FK520) by a combination of fluorescence, CD, FTIR and calorimetry. While only small changes in the overall structure of the protein may be induced by ligand, a large change in thermal stability of the binding protein is observed.     

Correlation between protein function and ligand binding profiles

We report that proteins with the same function bind the same set of small molecules from a standardized chemical library. This observation led to a quantifiable and rapidly adaptable method for protein functional analysis using experimentally derived ligand binding profiles. Ligand binding is measured using a high-throughput NMR ligand affinity screen with a structurally diverse chemical library. The method was demonstrated using a set of 19 proteins with a range of functions. A statistically significant similarity in ligand binding profiles was only observed between the two functionally identical albumins and between the five functionally similar amylases. This new approach is independent of sequence, structure, or evolutionary information and, therefore, extends our ability to analyze and functionally annotate novel genes.     

ASEdb: a database of alanine mutations and their effects on the free energy of binding in protein interactions

The Alanine Scanning Energetics database (ASEdb) is a searchable database of single alanine mutations in protein-protein, protein-nucleic acid, and protein-small molecule interactions for which binding affinities have been experimentally determined. In cases where structures are available, it contains surface areas of the mutated side chain and links to the PDB entries. It is useful for studying the contribution of single amino acids to the energetics of protein interactions, and can be updated by researchers as new data are generated.