Minor group human rhinovirus-receptor interactions: Geometry of multimodular attachment and basis of recognition

X-ray structures of human rhinovirus 2 (HRV2) in complex with soluble very-low-density lipoprotein receptors encompassing modules 1, 2, and 3 (V123) and five V3 modules arranged in tandem (V33333) demonstrates multi-modular binding around the virion’s five-fold axes. Occupancy was 60% for V123 and 100% for V33333 explaining the high-avidity of the interaction. Surface potentials of 3D-models of all minor group HRVs and K-type major group HRVs were compared; hydrophobic interactions between a conserved lysine in the viruses and a tryptophan in the receptor modules together with coulombic attraction via diffuse opposite surface potentials determine minor group HRV receptor specificity.     

The role of a conserved acidic residue in calcium-dependent protein folding for a low density lipoprotein (LDL)-A module: implications in structure and function for the LDL receptor superfamily

One common feature of the more than 1,000 complement-type repeats (or low density lipoprotein (LDL)-A modules) found in LDL receptor and the other members of the LDL receptor superfamily is a cluster of five highly conserved acidic residues in the C-terminal region, DXXXDXXDXXDE. However, the role of the third conserved aspartate of these LDL-A modules in protein folding and ligand recognition has not been elucidated. In this report, using a model LDL-A module and several experimental approaches, we demonstrate that this acidic residue, like the other four conserved acidic residues, is involved in calcium-dependent protein folding. These results suggest an alternative calcium coordination conformation for the LDL-A modules. The proposed model provides a plausible explanation for the conservation of this acidic residue among the LDL-A modules. Furthermore, the model can explain why mutations of this residue in human LDL receptor cause familial hypercholesterolemia.     

The crystal structure of a laminin G-like module reveals the molecular basis of alpha-dystroglycan binding to laminins, perlecan, and agrin

Laminin G-like (LG) modules in the extracellular matrix glycoproteins laminin, perlecan, and agrin mediate the binding to heparin and the cell surface receptor alpha-dystroglycan (alpha-DG). These interactions are crucial to basement membrane assembly, as well as muscle and nerve cell function. The crystal structure of the laminin alpha 2 chain LG5 module reveals a 14-stranded beta sandwich. A calcium ion is bound to one edge of the sandwich by conserved acidic residues and is surrounded by residues implicated in heparin and alpha-DG binding. A calcium-coordinated sulfate ion is suggested to mimic the binding of anionic oligosaccharides. The structure demonstrates a conserved function of the LG module in calcium-dependent lectin-like alpha-DG binding.     

Folding and binding integrity of variants of a prototype ligand-binding module from the LDL receptor possessing multiple alanine substitutions

The LA repeats that comprise the ligand-binding domain of the LDL receptor are among the most common autonomously structured extracellular modules found in the nonredundant protein sequence database. Here, we investigate the information content of the amino acid sequence of a typical LA module by constructing sequences with alanine residues at nonconserved positions in the module. Starting with the sequence of the fifth ligand-binding repeat of the LDL receptor (LA5), we created generic LA modules with alanine substitutions of nonconserved residues in only the N-terminal lobe, only the C-terminal lobe, and throughout both lobes of the module. LA variants with alanine residues at as many as 18 of 37 positions fold to a preferred disulfide isomer in the presence of calcium. Indeed, the six cysteines, the C-terminal calcium coordinating residues, two hydrophobic residues involved in packing, two glycines, and five other residues that form side chain-intramodule hydrogen bonds are alone sufficient to specify the fold of an LA module when alanine residues are present at all other positions. The LA variants with multiple alanines in either the N- or C-terminal lobe were then exploited to identify residues of LA5 that contribute to the binding of apoE-containing ligands in LDL receptor-derived "minireceptors", implicating nonconserved residues of the N-terminal lobe of LA5 in recognition of apoE-DMPC. Our library of LA modules with multiple alanine substitutions should be generally useful for probing the roles of nonconserved side chains in ligand recognition by proteins of the LDL receptor family.     

Structural independence of ligand-binding modules five and six of the LDL receptor

The low-density lipoprotein receptor (LDLR) is the primary mechanism for the uptake of plasma cholesterol into cells and serves as a prototype for a growing family of cell surface receptors. These receptors all utilize tandemly repeated LDL-A modules to bind their ligands. Each LDL-A module is about 40 residues long, has six conserved cysteine residues, and contains a conserved acidic region near the C-terminus which serves as a calcium-binding site. The structure of the interface presented for ligand binding by these modules, and the basis for their specificity and affinity in ligand binding, is not yet known. We have purified recombinant molecules corresponding to LDL-A modules five (LR5), six (LR6), and the module five-six pair (LR5-6) of the LDL receptor. Calcium is required to establish native disulfide bonds and to maintain the structural integrity of LR5, LR6, and the LR5-6 module pair. Folding studies of the I189D and D206Y mutations within LR5 indicate that each change leads to misfolding of the module, explaining the previous observation that each of these changes mimics the functional effect of deletion of the entire module [Russell, D. W., Brown, M. S., and Goldstein, J. L. (1989) J. Biol. Chem. 264, 21682-21688]. By fluorescence, the affinity of LR5 for calcium, which is crucial for folding and function of these modules, remains approximately 40 nM whether LR6 is attached. Comparison of proton and multidimensional heteronuclear NMR spectra of individual modules to those of the module pair indicates that most of the significant spectroscopic changes lie within the linker region between modules and that little structural interaction occurs between the cores of modules five and six in the 5-6 pair. These findings strongly support a model in which each module is essentially structurally independent of the other.     

The spacing between cysteines two and three of the LDL-A module of Tva is important for subgroup A avian sarcoma and leukosis virus entry

Rong et al. have demonstrated previously that with a few substitutions, the fourth repeat of human low-density lipoprotein (hLDL-A4) receptor can functionally replace the LDL-A module of Tva, the cellular receptor for subgroup A avian sarcoma and leukosis virus (ASLV-A), in viral entry (L. Rong, K. Gendron, and P. Bates, Proc. Natl. Acad. Sci. USA 95:8467-8472, 1998). Here we have shown that swapping the amino terminus of hLDL repeat 5 (hLDL-A5) with that of Tva, in addition to the corresponding substitutions made in human LDL-A4, was required to convert hLDL-A5 into an efficient ASLV-A receptor. These results substantiated our previous findings regarding the role of the specific residues in the viral interaction domain of Tva and demonstrated the critical role of the amino terminus of the Tva LDL-A module in ASLV-A infection. Furthermore, we have shown that the residues between cysteines 2 and 3 of the Tva LDL-A module in a Tva/LDL-A5 chimeric protein can be functionally replaced by the corresponding region of another LDL-A module, human LDL receptor-related protein repeat 22 (LDL-A22), to mediate efficient ASLV-A entry. Since the only conserved feature between the C2-C3 region of LDL-A22 and the Tva LDL-A module is that both contain nine amino acids of which none are conserved, we conclude that the spacing between C2 and C3 of the LDL-A module of Tva is an important determinant for ASLV-A entry. Thus, the present study provides strong evidence to support our hypothesis that one role of the N terminus of the LDL-A module of Tva is to allow proper folding and conformation of the protein for optimal interaction with the viral glycoprotein EnvA in ASLV-A entry.     

Solution structure of the viral receptor domain of Tva and its implications in viral entry

Tva is the cellular receptor for subgroup A avian sarcoma and leukosis virus (ASLV-A). The viral receptor function of Tva is determined by a 40-residue, cysteine-rich motif called the LDL-A module. Here we report the solution structure of the LDL-A module of Tva, determined by nuclear magnetic resonance (NMR) spectroscopy. Although the carboxyl terminus of the Tva LDL-A module has a structure similar to those of other reported LDL-A modules, the amino terminus adopts a different conformation. The LDL-A module of Tva does not contain the signature antiparallel beta-sheet observed in other LDL-A modules, and it is more flexible than other reported LDL-A modules. The LDL-A structure of Tva provides mechanistic insights into how a simple viral receptor functions in retrovirus entry. The side chains of H38 and W48 of Tva, which have been identified as viral contact residues by mutational analysis, are solvent exposed, suggesting that they are directly involved in EnvA binding. However, the side chain of L34, another potential viral contact residue identified previously, is buried inside of the module and forms the hydrophobic core with other residues. Thus L34 likely stabilizes the Tva structure but is not a viral interaction determinant. In addition, we propose that the flexible amino-terminal region of Tva plays an important role in determining specificity in the Tva-EnvA interaction.     

Sequence and structure of human rhinoviruses reveal the basis of receptor discrimination

The sequences of the capsid protein VP1 of all minor receptor group human rhinoviruses were determined. A phylogenetic analysis revealed that minor group HRVs were not more related to each other than to the nine major group HRVs whose sequences are known. Examination of the surface exposed amino acid residues of HRV1A and HRV2, whose X-ray structures are available, and that of three-dimensional models computed for the remaining eight minor group HRVs indicated a pattern of positively charged residues within the region, which, in HRV2, was shown to be the binding site of the very-low-density lipoprotein (VLDL) receptor. A lysine in the HI loop of VP1 (K224 in HRV2) is strictly conserved within the minor group. It lies in the middle of the footprint of a single repeat of the VLDL receptor on HRV2. Major group virus serotypes exhibit mostly negative charges at the corresponding positions and do not bind the negatively charged VLDL receptor, presumably because of charge repulsion.     

Solution structure of the sixth LDL-A module of the LDL receptor

The low-density lipoprotein receptor (LDLR) is the primary mechanism for uptake of plasma cholesterol into cells and serves as a prototype for an entire class of cell surface receptors. The amino-terminal domain of the receptor consists of seven LDL-A modules; the third through the seventh modules all contribute to the binding of low-density lipoproteins (LDLs). Here, we present the NMR solution structure of the sixth LDL-A module (LR6) from the ligand binding domain of the LDLR. This module, which has little recognizable secondary structure, retains the essential structural features observed in the crystal structure of LDL-A module five (LR5) of the LDLR. Three disulfide bonds, a pair of buried residues forming a hydrophobic "mini-core", and a calcium-binding site that serves to organize the C-terminal lobe of the module all occupy positions in LR6 similar to those observed in LR5. The striking presence of a conserved patch of negative surface electrostatic potential among LDL-A modules of known structure suggests that ligand recognition by these repeats is likely to be mediated in part by electrostatic complementarity of receptor and ligand. Two variants of LR6, identified originally as familial hypercholesterolemia (FH) mutations, have been investigated for their ability to form native disulfide bonds under conditions that permit disulfide exchange. The first, E219K, lies near the amino-terminal end of LR6, whereas the second, D245E, alters one of the aspartate side chains that directly coordinate the bound calcium ion. After equilibration at physiologic calcium concentrations, neither E219K nor D245E folds to a unique disulfide isomer, indicating that FH mutations both within and distant from the calcium-binding site give rise to protein-folding defects.     

Evidence of a potential receptor-binding site on the Nipah virus G protein (NiV-G): identification of globular head residues with a role in fusion promotion and their localization on an NiV-G structural model

As a preliminary to the localization of the receptor-binding site(s) on the Nipah virus (NiV) glycoprotein (NiV-G), we have undertaken the identification of NiV-G residues that play a role in fusion promotion. To achieve this, we have used two strategies. First, as NiV and Hendra virus (HeV) share a common receptor and their cellular tropism is similar, we hypothesized that residues functioning in receptor attachment could be conserved between their respective G proteins. Our initial strategy was to target charged residues (which can be expected to be at the surface of the protein) conserved between the NiV-G and HeV-G globular heads. Second, we generated NiV variants that escaped neutralization by anti-NiV-G monoclonal antibodies (MAbs) that neutralize NiV both in vitro and in vivo, likely by blocking receptor attachment. The sequencing of such "escape mutants" identified NiV-G residues present in the epitopes to which the neutralizing MAbs are directed. Residues identified via these two strategies whose mutation had an effect on fusion promotion were localized on a new structural model for the NiV-G protein. Our results suggest that seven NiV-G residues, including one (E533) that was identified using both strategies, form a contiguous site on the top of the globular head that is implicated in ephrinB2 binding. This site commences near the shallow depression in the center of the top surface of the globular head and extends to the rim of the barrel-like structure on the top loops of beta-sheet 5. The topology of this site is strikingly similar to that proposed to form the SLAM receptor site on another paramyxovirus attachment protein, that of the measles virus hemagglutinin.     

The Lin12-notch repeats of pregnancy-associated plasma protein-A bind calcium and determine its proteolytic specificity

The Lin12-Notch repeat (LNR) module of about 35 residues is a hallmark of the Notch receptor family. Three copies, arranged in tandem, are invariably present in the extracellular portion of the Notch receptors. Although their function is unknown, genetic and biochemical data indicate that the LNR modules participate in the regulation of ligand-induced proteolytic cleavage of the Notch receptor, a prerequisite to intramembrane cleavage and Notch signaling. Outside the Notch receptor family, the LNR module is present only in the metalloproteinase pregnancy-associated plasma protein-A (PAPP-A) and its homologue PAPP-A2, which also contain three copies. Curiously, LNR modules 1 and 2 are present within the proteolytic domain of PAPP-A/A2, but LNR3 is separated from LNR2 by more than 1000 amino acids. The growth factor antagonists insulin-like growth factor-binding protein (IGFBP)-4 and -5 are both substrates of PAPP-A. We provide here evidence that the PAPP-A LNR modules function together to determine the proteolytic specificity of PAPP-A. Analysis of C-terminally truncated PAPP-A mutants followed by the analysis of LNR deletion mutants demonstrated that each of the three PAPP-A LNR modules is strictly required for proteolytic activity against IGFBP-4 but not for proteolytic activity against IGFBP-5. Individual substitution of conserved LNR residues predicted to participate in calcium coordination caused elimination (D341A, D356A, D389A, D1484A, D1499A, and D1502A) or a significant reduction (D359A and E392A) of IGFBP-4 proteolysis, whereas IGFBP-5 proteolysis was unaffected. The activity of the latter mutants against IGFBP-4 could be partially rescued by calcium, and the addition of the calcium-binding protein calbindin D9k to wild-type PAPP-A eliminated activity against IGFBP-4 but not against IGFBP-5, demonstrating that the PAPP-A LNR modules bind calcium ions. We propose a model in which LNR3 is spatially localized in proximity to LNR1 and -2, forming a single functional unit.     

Modular construction of a tertiary RNA structure: the specificity domain of the Bacillus subtilis RNase P RNA

The structure of the specificity domain (S-domain) of the Bacillus subtilis RNase P RNA has been proposed to be composed of a core and a buttress module, analogous to the bipartite structure of the P4-P6 domain of the Tetrahymena group I ribozyme. The core module is the functional unit of the S-domain and contains the binding site for the T stem-loop of a tRNA. The buttress module provides structural stability to the core module and consists of a GA3 tetraloop and its receptor. To explicitly test the hypothesis that modular construction can describe the structure of the S-domain and is a useful RNA design strategy, we analyzed the equilibrium folding and substrate binding of three classes of S-domain mutants. Addition or deletion of a base pair in the helical linker region between the modules only modestly destabilizes the tertiary structure. tRNA binding selectivity is affected in one but not in two other mutants of this class. Elimination of the GA3 tetraloop-receptor interactions significantly destabilizes the core module and results in the loss of tRNA binding selectivity. Replacing the buttress module with that of a homologous RNase P RNA maintains the tRNA binding selectivity. Overall, we have observed that the linker regions between the two modules can tolerate moderate structural changes and that the buttress modules can be shuffled between homologous S-domains. These results suggest that it is feasible to design an RNA using a buttress module to stabilize a functional module.     

How does a DNA interacting enzyme change its specificity during molecular evolution? A site-directed mutagenesis study at the DNA binding site of the DNA-(adenine-N6)-methyltransferase EcoRV

The EcoRV DNA-(adenine-N6)-methyltransferase (MTase) recognizes GATATC sequences and modifies the first adenine residue within this site. Parts of its DNA interface show high sequence homology to DNA MTases of the dam family which recognize and modify GATC sequences. A phylogenetic analysis of M.EcoRV and dam-MTases suggests that EcoRV arose in evolution from a primordial dam-MTase in agreement to the finding that M.EcoRV also methylates GATC sites albeit at a strongly reduced rate. GATCTC sites that deviate in only one position from the EcoRV sequence are preferred over general dam sites. We have investigated by site-directed mutagenesis the function of 17 conserved and nonconserved residues within three loops flanking the DNA binding cleft of M.EcoRV. M.EcoRV contacts the GATATC sequence with two highly cooperative recognition modules. The contacts to the GAT-part of the recognition sequence are formed by residues conserved between dam MTases and M.EcoRV. Mutations at these positions lead to an increase in the discrimination between GATATC and GATC substrates. Our data show that the change in sequence specificity from dam (GATC) to EcoRV (GATATC) was accompanied by the generation of a second recognition module that contacts the second half of the target sequence. The new DNA contacts are formed by residues from all three loops that are not conserved between M.EcoRV and dam MTases. Mutagenesis at important residues within this module leads to variants that show a decreased ability to recognize the TC-part of the GATATC sequence.     

Crystal structure of the third extracellular domain of CD5 reveals the fold of a group B scavenger cysteine-rich receptor domain

Scavenger receptor cysteine-rich (SRCR) domains are ancient protein modules widely found among cell surface and secreted proteins of the innate and adaptive immune system, where they mediate ligand binding. We have solved the crystal structure at 2.2 A of resolution of the SRCR CD5 domain III, a human lymphocyte receptor involved in the modulation of antigen specific receptor-mediated T cell activation and differentiation signals. The first structure of a member of a group B SRCR domain reveals the fold of this ancient protein module into a central core formed by two antiparallel beta-sheets and one alpha-helix, illustrating the conserved core at the protein level of genes coding for group A and B members of the SRCR superfamily. The novel SRCR group B structure permits the interpretation of site-directed mutagenesis data on the binding of activated leukocyte cell adhesion molecule (ALCAM/CD166) binding to CD6, a closely related lymphocyte receptor homologue to CD5.     

Molecular engineering of a thermostable carbohydrate-binding module

Structure-function studies are frequently practiced on the very diverse group of natural carbohydrate-binding modules in order to understand the target recognition of these proteins. We have taken a step further in the study of carbohydrate-binding modules and created variants with novel binding properties by molecular engineering of one such molecule of known 3D-structure. A combinatorial library was created from the sequence encoding a thermostable carbohydrate-binding module, CBM4-2 from a Rhodothermus marinus xylanase, and the phage-display technology was successfully used for selection of variants with specificity towards different carbohydrate polymers (birchwood xylan, Avicel™, ivory nut mannan and recently also xyloglucan), as well as towards a glycoprotein (human IgG4). Our work not only generated a number of binders with properties that would suite a range of biotechnological applications, but analysis the selected binders also helped us to identify residues important for their specificities.     

Predictive bioinformatic identification of minor receptor group human rhinoviruses

Major group HRVs bind intercellular adhesion molecule 1 and minor group HRVs bind members of the low-density lipoprotein receptor (LDLR) family for cell entry. Whereas the former share common sequence motives in their viral capsid proteins (VPs), in the latter only a lysine residue within the binding epitope in VP1 is conserved; this lysine is also present in “K-type” major group HRVs that fail to use LDLR for infection. By using the available sequences three-dimensional models of VP1 of all HRVs were built and binding energies, with respect to module 3 of the very-low-density lipoprotein receptor, were calculated. Based on the predicted affinities K-type HRVs and minor group HRVs were correctly classified.     

Molecular engineering of a thermostable carbohydrate-binding module

Structure–function studies are frequently practiced on the very diverse group of natural carbohydrate-binding modules in order to understand the target recognition of these proteins. We have taken a step further in the study of carbohydrate-binding modules and created variants with novel binding properties by molecular engineering of one such molecule of known 3D-structure. A combinatorial library was created from the sequence encoding a thermostable carbohydrate-binding module, CBM4-2 from a Rhodothermus marinus xylanase, and the phage-display technology was successfully used for selection of variants with specificity towards different carbohydrate polymers (birchwood xylan, Avicel?, ivory nut mannan and recently also xyloglucan), as well as towards a glycoprotein (human IgG4). Our work not only generated a number of binders with properties that would suite a range of biotechnological applications, but analysis the selected binders also helped us to identify residues important for their specificities.     

Mapping the hyaluronan-binding site on the link module from human tumor necrosis factor-stimulated gene-6 by site-directed mutagenesis

Link modules are hyaluronan-binding domains found in extracellular proteins involved in matrix assembly, development, and immune cell migration. Previously we have expressed the Link module from the inflammation-associated protein tumor necrosis factor-stimulated gene-6 (TSG-6) and determined its tertiary structure in solution. Here we generated 21 Link module mutants, and these were analyzed by nuclear magnetic resonance spectroscopy and a hyaluronan-binding assay. The individual mutation of five amino acids, which form a cluster on one face of the Link module, caused large reductions in functional activity but did not affect the Link module fold. This ligand-binding site in TSG-6 is similar to that determined previously for the hyaluronan receptor, CD44, suggesting that the location of the interaction surfaces may also be conserved in other Link module-containing proteins. Analysis of the sequences of TSG-6 and CD44 indicates that the molecular details of their association with hyaluronan are likely to be significantly different. This comparison identifies key sequence positions that may be important in mediating hyaluronan binding, across the Link module superfamily. The use of multiple sequence alignment and molecular modeling allowed the prediction of functional residues in link protein, and this approach can be extended to all members of the superfamily.