Structure,stability and dynamics of the central domain of cardiac myosin binding protein C (MyBP-C): implications for multidomain assembly and causes for cardiomyopathy

The large multidomain muscle protein myosin binding protein C (MyBP-C) has been implicated for some time in cardiac disease while until recently little was known about its structure and function. Here we present a detailed study of the central domain C5 of the cardiac isoform of MyBP-C. This domain is unusual in several aspects. Firstly it contains two sizeable insertions compared to the non-cardiac isoforms. The first insertion comprises the linker between domains cC4 and cC5 that is elongated by ten amino acid residues, the second insertion comprises an elongation of the CD-loop in the middle of the domain by approximately 30 amino acid residues. Secondly two point mutations linked to familial hypertrophic cardiomyopathy (FHC) have been identified in this domain. This work shows that the general fold of cC5 is in agreement with the IgI family of beta-sandwich structures. The long cardiac-specific linker between cC4 and cC5 is not a linker at all but an integral part of the fold of cC5, as evidenced by an unfolded mutant in which this segment was removed. The second insertion is shown to be unstructured, highly dynamic and mostly extended according to NMR relaxation measurements and analytical ultracentrifugation. The loss of several key interactions conserved in the CD-loop of the IgI fold is assumed to be responsible for the low stability of cC5 compared to other IgI domains from titin and MyBP-C itself. The low thermodynamic stability of cC5 is most evident in one of the two FHC-linked mutations, N755K (Asn115 in this construct) which is mainly unfolded with a small proportion of a native-like folded species. In contrast, the second FHC-linked mutation, R654H (Arg14 in this construct) is as well folded and stable as the wild-type. This residue is located in the extended beta-bulge at the N terminus of the protein, pointing towards the surface of the CFGA' beta-sheet. This position is in agreement with recent data pointing to a function of Arg654 in an intermolecular interaction with MyBP-C domain cC8.     

Analyzing pathogenic mutations of C5 domain from cardiac myosin binding protein C through MD simulations

The folding properties of wild type and mutants of domain C5 from cardiac myosin binding protein C have been investigated via molecular dynamics simulations within the framework of a native-centric and coarse-grained model. The relevance of a mutation has been assessed through the shift in the unfolding temperature, the change in the unfolding rate it determines and Phi-values analysis. In a previous paper (Guardiani et al. Biophys J 94:1403-1411, 2008), we performed Kinetic simulations on native contact formation revealing an entropy-driven folding pathway originating near the FG and DE loops. This folding mechanism allowed also a possible interpretation of the molecular impact of the three mutations, Arg14His, Arg28His and Asn115Lys involved in the Familial Hypertrophic Cardiomyopathy. Here we extend that analysis by enriching the mutant pool and we identify a correlation between unfolding rates and the number of native contacts retained in the transition state.     

Stability and kinetic properties of C5-domain from myosin binding protein C and its mutants

The impact of three mutations of domain C5 from myosin binding protein C, correlated to Familial Hypertrophic Cardiomyopathy, has been assessed through molecular dynamics simulations based on a native centric protein modeling. The severity of the phenotype correlates with the shift in unfolding temperature determined by the mutations. A contact probability analysis reveals a folding process of the C5 domain originating in the region of DE and FG loops and propagating toward the area proximal to CD and EF loops. This suggests that mutation effects gain relevance in the proximity to the area where folding originates. The scenario is also confirmed by the analysis of the kinetics of 27 test mutations evenly distributed throughout the entire C5 domain.     

Impact of mutations in nucleoprotein on replication of influenza virus A/Hong Kong/1/68/162/35 reassortants at different temperatures

The nucleoprotein (NP) of influenza virus is a multifunctional RNA binding protein. The role of NP in the adaptation of influenza viruses to a host has been experimentally proved. Ambiguous data are available on the role of nucleoprotein in the attenuation of influenza A viruses, which is characterized by ability to replicate at low temperature (26°C) and inability to replicate at high temperature (39°C). Influenza virus donor strain A/Hong Kong/1/68/162/35 (H3N2), adapted to growth at low temperature, differs from the wild type virus by 14 amino acid mutations in the internal and non-structural proteins. Two mutations occurred in the NP: Gly102Arg and Glu292Gly. We have obtained viruses with point reverse-mutations in these positions and compared their replication at different temperatures by measuring infectious activity in chicken embryos. It has been shown that reverse mutation Gly292Glu in the NP reduced virus ability to replicate at low temperature, the introduction of the second reverse mutation Arg102Gly completely abolished virus cold adaptation.     

NMR structure of the LCCL domain and implications for DFNA9 deafness disorder

The LCCL domain is a recently discovered, conserved protein module named after its presence in Limulus factor C, cochlear protein Coch-5b2 and late gestation lung protein Lgl1. The LCCL domain plays a key role in the autosomal dominant human deafness disorder DFNA9. Here we report the nuclear magnetic resonance (NMR) structure of the LCCL domain from human Coch-5b2, where dominant mutations leading to DFNA9 deafness disorder have been identified. The fold is novel. Four of the five known DFNA9 mutations are shown to involve at least partially solvent-exposed residues. Except for the Trp91Arg mutant, expression of these four LCCL mutants resulted in misfolded proteins. These results suggest that Trp91 participates in the interaction with a binding partner. The unexpected sensitivity of the fold with respect to mutations of solvent-accessible residues might be attributed to interference with the folding pathway of this disulfide-containing domain.     

Structural definition of the C5a C terminus by two-dimensional nuclear magnetic resonance spectroscopy

The serum glycoprotein C5a, which is derived from the proteolytic cleavage of complement protein C5, has been implicated in the pathogenesis of a number of inflammatory and allergic conditions. Because C5a induces an inflammatory response upon binding to a specific receptor, structural and mutagenesis studies were carried out to gain a better understanding of this binding interaction. These studies led to the first structural definition of the C terminus of recombinant human (rh)-C5a, determined by two-dimensional nuclear magnetic resonance (NMR) spectroscopy. Our results show that the C terminus adopts an α-helical conformation spanning residues 69 to 74, while the core domain exists as an antiparallel α-helical bundle. This C-terminal helix is connected to the core by a short loop that orients Arg 74 adjacent to Arg 62. Point mutation analysis had already revealed that residues 62 and 74 significantly contribute to agonist activity and receptor binding. Correlation of the C5a tertiary structure with mutational analyses clarifies the significance of the functional and binding properties of Arg 62 and suggests that both Arg 62 and Arg 74 interact at the same binding site on the receptor. Proteins 28:261–267, 1997 © 1997 Wiley-Liss Inc.     

Congenital insulin resistance associated with a conformational alteration in a conserved beta-sheet in the insulin receptor L1 domain.

The hormone binding site of members of the insulin receptor family is contained within a highly conserved extracellular region of the receptor. Recent crystallization of the N-terminal region of the binding site revealed two large domains (L1, L2), each organized as a single-stranded right-handed beta-helix, connected by a rod-shaped cysteine-rich domain. Here, we analyze two new naturally occurring mutations in a single beta-sheet within L1, D59G and L62P, that we previously identified in a young woman with classic congenital insulin resistance (type A). Substitution of D59G, a beta-sheet connecting loop residue, caused decreased hormone binding but did not disrupt overall folding, assembly, or movement to the cell surface. In contrast, replacement of the adjacent residue L62P, which is located within the beta-sheet, and positioned in a hormone binding surface, completely disrupted intracellular folding, oligomerization, and trafficking and resulted in aberrant proteolytic degradation. Immunohistochemistry in combination with biosynthetic studies showed that misfolded receptors were retained in an incorrect cellular location and that they colocalized with the resident endoplasmic reticulum chaperone calnexin. This study, together with other mutagenesis data, shows that formation of beta-sheet elements within the L1 beta-helix are critical for the folding of the entire extracellular domain of the receptor and that the hormone contact site is composed in part by residues in this domain.     

Localization of von willebrand factor-binding sites for platelet glycoprotein Ib and botrocetin by charged-to-alanine scanning mutagenesis

At sites of vascular injury, von Willebrand factor (VWF) mediates platelet adhesion through binding to platelet glycoprotein Ib (GPIb). Previous studies identified clusters of charged residues within VWF domain A1 that were involved in binding GPIb or botrocetin. The contribution of 28 specific residues within these clusters was analyzed by mutating single amino acids to alanine. Binding to a panel of six conformation-dependent monoclonal antibodies was decreased by mutations at Asp(514), Asp(520), Arg(552), and Arg(611) (numbered from the N-terminal Ser of the mature processed VWF), suggesting that these residues are necessary for domain A1 folding. Binding of (125)I-botrocetin was decreased by mutations at Arg(629), Arg(632), Arg(636), and Lys(667). Ristocetin-induced and botrocetin-induced binding to GPIb both were decreased by mutations at Lys(599), Arg(629), and Arg(632); among this group the K599A mutant was unique because (125)I-botrocetin binding was normal, suggesting that Lys(599) interacts directly with GPIb. Ristocetin and botrocetin actions on VWF were dissociated readily by mutagenesis. Ristocetin-induced binding to GPIb was reduced selectively by substitutions at positions Lys(534), Arg(571), Lys(572), Glu(596), Glu(613), Arg(616), Glu(626), and Lys(642), whereas botrocetin-induced binding to GPIb was decreased selectively by mutations at Arg(636) and Lys(667). The binding of monoclonal antibody B724 involved Lys(660) and Arg(663), and this antibody inhibits (125)I-botrocetin binding to VWF. The crystal structure of the A1 domain suggests that the botrocetin-binding site overlaps the monoclonal antibody B724 epitope on helix 5 and spans helices 4 and 5. The binding of botrocetin also activates the nearby VWF-binding site for GPIb that involves Lys(599) on helix 3.     

Chemotactic responses of human peripheral blood monocytes to the complement-derived peptides C5a and C5a des Arg

We examined responses of human peripheral blood polymorphonuclear leukocytes (PMN) and monocytes to the highly purified human complement-derived peptides C5a and C5a des Arg. As reported previously, C5a proved to be approximately 10- to 20-fold more potent than C5a des Arg as a chemoattractant for human PMN. C5a also was more potent than C5a des Arg in causing PMN to acquire a polarized morphology. In contrast, we found that human monocytes do not distinguish between C5a and C5a des Arg when these peptides are used as chemoattractants. In two different assay systems, both peptides acted at identical concentrations to stimulate suboptimal and optimal migration of monocytes. Human monocytes also did not distinguish between C5a and C5a des Arg when these peptides were used as inducers of polarization. Studies performed with functionally active, [125I]-labeled C5a and C5a des Arg, however, demonstrated that binding of C5a des Arg to monocytes differed from binding of C5a. Although [125I]-C5a des Arg appeared to bind to the same receptor as [125I]-C5a, binding of labeled C5a des Arg occurred with an affinity that was approximately 100-fold less than that observed with labeled C5a. These results indicate that leukocyte chemotactic and polarization responses to C5a and C5a des Arg vary, depending on the target cell type.     

The role of the N-terminal domain of the complement fragment receptor C5L2 in ligand binding

C5L2 is a new cellular receptor found to interact with the human anaphylatoxins complement factor C5a and its C-terminal cleavage product C5a des Arg. The classical human C5a receptor (C5aR) preferentially binds C5a, with a 10-100-fold lower affinity for C5a des Arg. In contrast, C5L2 binds both ligands with nearly equal affinity. C5aR presents acidic and tyrosine residues in its N terminus that interact with the core of C5a while a hydrophobic pocket formed by the transmembrane helices interacts with residues in the C terminus of C5a. Here, we have investigated the molecular basis for the increased affinity of C5L2 for C5a des Arg. Rat and mouse C5L2 preferentially bound C5a des Arg, whereas rodent C5aR showed much higher affinity for intact C5a. Effective peptidic and non-peptidic ligands for the transmembrane hydrophobic pocket of C5aR were poor inhibitors of ligand binding to C5L2. An antibody raised against the N terminus of human C5L2 did not affect the binding of C5a to C5L2 but did inhibit C5a des Arg binding. A chimeric C5L2, containing the N terminus of C5aR, had little effect on the affinity for C5a des Arg. Mutation of acidic and tyrosine residues in the N terminus of human C5L2 revealed that 3 residues were critical for C5a des Arg binding but had little involvement in C5a binding. C5L2 thus appears to bind C5a and C5a des Arg by different mechanisms, and, unlike C5aR, C5L2 uses critical residues in its N-terminal domain for binding only to C5a des Arg.     

Molecular Modeling of Disease Causing Mutations in Domain C1 of cMyBP-C

Cardiac myosin binding protein-C (cMyBP-C) is a multi-domain (C0–C10) protein that regulates heart muscle contraction through interaction with myosin, actin and other sarcomeric proteins. Several mutations of this protein cause familial hypertrophic cardiomyopathy (HCM). Domain C1 of cMyBP-C plays a central role in protein interactions with actin and myosin. Here, we studied structure-function relationship of three disease causing mutations, Arg177His, Ala216Thr and Glu258Lys of the domain C1 using computational biology techniques with its available X-ray crystal structure. The results suggest that each mutation could affect structural properties of the domain C1, and hence it’s structural integrity through modifying intra-molecular arrangements in a distinct mode. The mutations also change surface charge distributions, which could impact the binding of C1 with other sarcomeric proteins thereby affecting contractile function. These structural consequences of the C1 mutants could be valuable to understand the molecular mechanisms for the disease.     

Structural bases of the redox-dependent conformational switch in the serpin PAI-2

Depending on the redox-status, the serpin plasminogen activator inhibitor type 2 (PAI-2) can exist in either a stable monomeric or polymerogenic form. The latter form, which spontaneously forms loop-sheet polymers, has an open beta-sheet A and is stabilized by a disulfide bond between C79 (in the CD-loop) and C161 (at the bottom of PAI-2). Reduction of this bond results in a closing of the beta-sheet A and converts PAI-2 to a stable monomeric form. Here we show that the stable monomeric and polymerogenic forms of PAI-2 are fully interconvertible, depending on redox-status of the environment. Our intramolecular distance measurements indicate that the CD-loop folds mainly on one side of the stable monomeric form of the inhibitor. However, the loop can translocate about 54A to the bottom of PAI-2 so that the C79-C161 disulfide bond can form under oxidizing conditions. We show also that the redox-active C79 can form a disulfide-link to the matrix protein vitronectin, suggesting that vitronectin can stabilize active PAI-2 in extracellular compartments. PAI-2 is therefore a rare example of a redox-sensitive protein for which the activity and polymerization ability are regulated by reversible disulfide bond formation leading to major translocation of a loop and significant conformational changes in the molecule.     

Activated protein C-protein C inhibitor complex formation: characterization of a neoepitope provides evidence for extensive insertion of the reactive center loop

Protein C inhibitor, a serine proteinase inhibitor (serpin), is the physiologically most important inhibitor of activated protein C. We have made a monoclonal antibody (M36) that binds with equally high affinity to an epitope present in activated protein C-protein C inhibitor complexes and cleaved loop-inserted protein C inhibitor. Insertion of a synthetic N-acetylated tetradecapeptide (corresponding to residues P1-P14 of the reactive center loop) into beta-sheet A of the uncleaved inhibitor also exposed the epitope. The antibody had no apparent affinity for native uncleaved inhibitor or for the free peptide. Synthetic P1-P14 analogues, with Arg P13 or Ala P9 substituted to the residues found in mouse protein C inhibitor (Thr and Ile, respectively), were also inserted in beta-sheet A. The Arg P13/Thr substitution led to a greatly impaired reactivity with the antibody, whereas the Ala P9/Ile mutation resulted in a modest loss of reactivity with the antibody. These results indicate that complex formation leads to insertion of the reactive center loop in beta-sheet A from Arg P14 and presumably beyond Ala P9. Moreover, to the best of our knowledge, this is the first instance where the neoepitope of a complexation-specific monoclonal antibody has been localized to the loop-inserted part of beta-sheet A, the part of the serpin where the complexation-induced conformational change is most conspicuous.     

Involvement of the N- and C-terminal fragments of bovine pancreatic deoxyribonuclease in active protein folding

The three-dimensional structure of bovine pancreatic (bp) DNase revealed that its N- and C-termini form an antiparallel beta-sheet structure. The involvement of this beta-sheet structure in the active protein folding of bpDNase was thus investigated via a series of deletion and substitution variants. Several substitution variants of N-terminal Leu1 and C-terminal Leu259, and one variant with only the last Thr260 deleted, remained fully active. However, the other deletion variants, in which 2-10 amino acid residues were removed from the C- or N-terminus, all lost the DNase activity. The results indicated that the backbone hydrogen bonding in the antiparallel beta-sheet, rather than the side-chain interactions, is crucial for the correct protein folding. When the deletion variants were complemented with synthetic peptides of the deleted N- or C-terminal sequences, the DNase activity was generated. The highest DNase activity was generated when the C-terminal 10-residue-deleted brDNase(Delta251-260) was admixed with the C-terminal 10-residue peptide (peptide C10) in a molar ratio of 1:400. The noncovalent binding between brDNase(Delta251-260) and peptide C10 exhibited a dissociation constant of 48 microM. Circular dichroism spectra showed that the deletion variants were partially folded with mainly helical structures and that admixture with corresponding peptides facilitated their folding into the nativelike beta-sheet-rich structure. Thermal denaturation profiles also revealed that the transition temperature for brDNase(Delta251-260) was increased from 55 to 63 degrees C after incubation with peptide C10. The folding activation process for the deletion variant occurred in two stages, and Ca(2+) was required.     

Mutational hotspots in electron transfer flavoprotein underlie defective folding and function in multiple acyl-CoA dehydrogenase deficiency

We have carried out an extensive in silico analysis on 18 disease associated missense mutations found in electron transfer flavoprotein (ETF), and found that mutations fall essentially in two groups, one in which mutations affect protein folding and assembly, and another one in which mutations impair catalytic activity and disrupt interactions with partner dehydrogenases. We have further experimentally analyzed three of these mutations, ETFβ-p.Cys42Arg, ETFβ-p.Asp128Asn and ETFβ–p.Arg191Cys, which have been found in homozygous form in patients and which typify different scenarios in respect to the clinical phenotypes. The ETFβ-p.Cys42Arg mutation, related to a severe form of multiple acyl-CoA dehydrogenase deficiency (MADD), affects directly the AMP binding site and intersubunit contacts and impairs correct protein folding. The two other variations, ETFβ-p.Asp128Asn and ETFβ–p.Arg191Cys, are both associated with mild MADD, but these mutations have a different impact on ETF. Although none affects the overall α/β fold topology as shown by far-UV CD, analysis of the purified proteins shows that both have substantially decreased enzymatic activity and conformational stability. Altogether, this study combines in silico analysis of mutations with experimental data and has allowed establishing structural hotspots within the ETF fold that are useful to provide a rationale for the prediction of effects of mutations in ETF.     

A breakdown of symmetry in the folding transition state of protein L

The 62 residue IgG binding domain of protein L consists of a central alpha-helix packed on a four-stranded beta-sheet formed by N and C-terminal beta-hairpins. The overall topology of the protein is quite symmetric: the beta-hairpins have similar lengths and make very similar interactions with the central helix. Characterization of the effects of 70 point mutations distributed throughout the protein on the kinetics of folding and unfolding reveals that this symmetry is completely broken during folding; the first beta-hairpin is largely structured while the second beta-hairpin and helix are largely disrupted in the folding transition state ensemble. The results are not consistent with a "hydrophobic core first" picture of protein folding; the first beta-hairpin appears to be at least as ordered at the rate limiting step in folding as the hydrophobic core.     

In silico Investigation of the Disease-Associated Retinoschisin C110Y and C219G Mutants

The juvenile X-linked retinoschisis (XLRS) is a retinal disease caused by mutations in the secretory protein, retinoschisin (RS1). Majority of the disease is resulted from single point mutations on the RS1 discoidin domain with cysteine mutations being related to some of the more severe cases of XLRS. Previous studies have indicated that two mutations (C110Y and C219G), which involve cysteines that form intramolecular disulfide bonds in the native discoidin domain, resulted in different oligomerization states of the proteins and did not correlate with the degree of protein stability as calculated by the change in folding free energy. Through homology modeling, bioinformatics predictions, molecular dynamics (MD) and docking simulations, we attempt to investigate the effects of these two mutations on the structure of the RS1 discoidin domain in relevance to the discrepancy found between structural stability and aggregation propensity. Based on our findings, this discrepancy can be explained by the ability of C110Y mutant to establish suitable modules for initiating amorphous aggregation and to expand the aggregating mass through predominantly hydrophobic interactions. The low capability of C219G mutant to oligomerize, on the other hand, may be due to its greater structural instability and lesser hydrophobic tendency, two properties that may be unsupportive of aggregation. The results, altogether, indicate that aggregation propensity in the RS1 C110Y mutant is dependent upon the formation of suitable aggregating substrates for propagation of aggregation and not directly related to or determined by overall structural instability. As for the wildtype protein, the binding specificity of the spikes for biological function and the formation of octameric structure are contributed by important loop interactions, as well as evolved structural and sequence-based properties that prevent aggregation.     

A La protein requirement for efficient pre-tRNA folding

The La protein protects the 3' ends of many nascent small RNAs from exonucleases. Here we report that La is required for efficient folding of certain pre-tRNAs. A mutation in pre-tRNA(Arg)(CCG) causes yeast cells to be cold-sensitive and to require the La protein Lhp1p for efficient growth. When the mutant cells are grown at low temperature, or when Lhp1p is depleted, mature tRNA(Arg)(CCG) is not efficiently aminoacylated. The mutation causes the anticodon stem of pre-tRNA(Arg)(CCG) to misfold into an alternative helix in vitro. Intragenic suppressor mutations that disrupt the misfolded helix or strengthen the correct helix alleviate the requirement for Lhp1p, providing evidence that the anticodon stem misfolds in vivo. Chemical and enzymatic footprinting experiments suggest a model in which Lhp1p stabilizes the correctly folded stem. Lhp1p is also required for efficient aminoacylation of two wild-type tRNAs when yeast are grown at low temperature. These experiments reveal that pre-tRNAs can require protein assistance for efficient folding in vivo.     

Identification of functionally important amino acid residues within the C2-domain of human factor V using alanine-scanning mutagenesis

We have previously determined that the C2-domain of human factor V (residues 2037-2196) is required for expression of cofactor activity and binding to phosphatidylserine (PS)-containing membranes. Naturally occurring factor V inhibitors and a monoclonal antibody (HV-1) recognized epitopes in the amino terminus of the C2-domain (residues 2037-2087) and blocked PS binding. We have now investigated the function of individual amino acids within the C2-domain using charge to alanine mutagenesis. Charged residues located within the C2-domain were changed to alanine in clusters of 1-3 mutations per construct. In addition, mutants W2063A, W2064A, (W2063, W2064)A, and L2116A were constructed as well. The resultant 30 mutants were expressed in COS cells using a B-domain deleted factor V construct (rHFV des B). All mutants were expressed efficiently based on the polyclonal antibody ELISA. The charged residues, Arg(2074), Asp(2098), Arg(2171), Arg(2174), and Glu(2189) are required for maintaining the structural integrity of the C2-domain of factor V. Four of these residues (Arg(2074), Asp(2098), Arg(2171), and Arg(2174)) correspond to positions in the factor VIII C-type domains that have been identified as point mutations in patients with hemophilia A. The epitope for the inhibitory monoclonal antibody HV-1 has been localized to Lys(2060) through Glu(2069) in the factor V C2-domain. The epitope for the inhibitory monoclonal antibody 6A5 is composed of amino acids His(2128) through Lys(2137). The PS-binding site in the factor V C2-domain includes amino acid residues Trp(2063) and Trp(2064). This site overlaps with the epitope for monoclonal antibody HV-1. These factor V C2-domain mutants should provide valuable tools for further defining the molecular interactions responsible for factor V binding to phospholipid membranes.     

Characterization of the role of the N-loop of MIP-1 beta in CCR5 binding

MIP-1beta is a CC-chemokine that plays a role in inflammation and host defense mechanisms by interacting with its specific receptor CCR5. CCR5 is a major coreceptor for macrophage-tropic human immunodeficiency virus (HIV), and as a consequence, MIP-1beta can inhibit HIV entry. It is therefore of interest to understand how MIP-1beta and other CCR5 ligands bind to their receptor, as such understanding could lead to the rational design of more efficient HIV entry blockers. We have previously demonstrated the importance of Phe13, and of basic residues of the 40's loop, in mediating high-affinity binding of MIP-1beta to CCR5. We have now investigated further the relative contribution of other MIP-1beta residues in the interaction of the chemokine with CCR5, by studying the functional consequences of point mutations within the N-loop and the 3(10) turn of MIP-1beta, affecting the charge, size, and H-bonding properties of the side chains. Our data suggest that, in addition to Phe13, three amino acids of the N-loop and 3(10) turn (Arg18, Lys19, and Arg22) interact with CCR5 through their positive charge. We also found that Pro21 contributes to the CCR5 binding properties of MIP-1beta. Moreover, NMR spectroscopy has revealed that the presence of Tyr at position 15 is necessary for the proper folding of the chemokine. Our results therefore demonstrate that the binding determinants of MIP-1beta consist of residues arranged on one surface of the protein, including most of the basic residues in MIP-1beta, as well as two key hydrophobic groups. The good correlation observed between the potency of the mutants in a functional assay and their binding affinity strongly argues that basic residues Arg18, Lys19, and Arg22 of MIP-1beta are essential for its CCR5 binding properties, without a primary effect on CCR5 activation.