Proteins evolve in a fitness landscape encompassing a complex network of biological constraints. Because of the interrelation of folding, function, and regulation, the ground-state structure of a ...protein may be inactive. A model is provided by insulin, a vertebrate hormone central to the control of metabolism. Whereas native assembly mediates storage within pancreatic β-cells, the active conformation of insulin and its mode of receptor binding remain elusive. Here, functional surfaces of insulin were probed by photocross-linking of an extensive set of azido derivatives constructed by chemical synthesis. Contacts are circumferential, suggesting that insulin is encaged within its receptor. Mapping of photoproducts to the hormone-binding domains of the insulin receptor demonstrated alternating contacts by the B-chain β-strand (residues B24-B28). Whereas even-numbered probes (at positions B24 and B26) contact the N-terminal L1 domain of the α-subunit, odd-numbered probes (at positions B25 and B27) contact its C-terminal insert domain. This alternation corresponds to the canonical structure of aβ-strand (wherein successive residues project in opposite directions) and so suggests that the B-chain inserts between receptor domains. Detachment of a receptor-binding arm enables photo engagement of surfaces otherwise hidden in the free hormone. The arm and associated surfaces contain sites also required for nascent folding and self-assembly of storage hexamers. The marked compression of structural information within a short polypeptide sequence rationalizes the diversity of diabetes-associated mutations in the insulin gene. Our studies demonstrate that photoscanning mutagenesis can decode the active conformation of a protein and so illuminate cryptic constraints underlying its evolution.
Diabetes-Associated Mutations in Insulin Identify Invariant Receptor Contacts
Bin Xu 1 ,
Shi-Quan Hu 2 ,
Ying-Chi Chu 2 ,
Shuhua Wang 2 ,
Run-ying Wang 2 ,
Satoe H. Nakagawa 3 ,
Panayotis G. ...Katsoyannis 2 and
Michael A. Weiss 1
1 Department of Biochemistry, Case Western Reserve School of Medicine, Cleveland, Ohio
2 Department of Pharmacology and Biological Chemistry, Mount Sinai School of Medicine, New York University, New York, New York
3 Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois
Address correspondence and reprint requests to Michael A. Weiss, Case Western Reserve University, Department of Biochemistry,
10900 Euclid Ave., SOM Room W427, Cleveland, OH 44106-4935. E-mail: michael.weiss{at}case.edu
Abstract
Mutations in human insulin cause an autosomal-dominant syndrome of diabetes and fasting hyperinsulinemia. We demonstrate by
residue-specific photo cross-linking that diabetes-associated mutations occur at receptor-binding sites. The studies use para -azido-phenylalanine, introduced at five sites by total protein synthesis. Because two such sites (Val A3 and Phe B24 ) are largely buried in crystal structures of the free hormone, their participation in receptor binding is likely to require
a conformational change to expose a hidden functional surface. Our results demonstrate that this surface spans both chains
of the insulin molecule and includes sites of rare human mutations that cause diabetes.
DTT, dithiothreitol
FnIII1, second fibronectin-homology domain
ID, insert domain
Pap, para-azido-phenylalanine
Phe, phenylalanine
Footnotes
Accepted February 25, 2004.
Received December 11, 2003.
DIABETES
We performed a mesocosms experiment using a vertical-flow wetland system to treat liquid sludge in the Mediterranean region. Three common helophyte species, common reed (
Phragmites australis Cav.), ...broadleaf cattail (
Typha latifolia L.), and yellow flag (
Iris pseudacorus L.), were planted as monoculture and irrigated with a liquid sewage sludge from a food industry, characterised by very high organic concentrations (COD
>
8000
mg/L). We studied the benefits of plants by comparing unplanted to planted mesocosms. Results showed the high performance of such vertical-flow wetland systems. Removal efficiency was more than 98% for total suspended solids (TSS) and chemical oxygen demand (COD), and more than 87% for total Kjeldahl nitrogen (TKN). The main removal process was physical filtration by the substrate due to the high proportion of particulate elements in the sludge. Planted mesocosms were more efficient than those unplanted, confirming the positive role of the plants. Mesocosms planted with
Phragmites or
Typha showed better performances in TKN removal than those planted with
Iris. Only in mesocosms planted with
Phragmites was there no outflow in summer due to high evapotranspiration.
The contribution of the insulin A-chain to receptor binding is investigated by photo-cross-linking and nonstandard mutagenesis. Studies focus on the role of ValA³, which projects within a crevice ...between the A- and B-chains. Engineered receptor α-subunits containing specific protease sites ("midi-receptors") are employed to map the site of photo-cross-linking by an analog containing a photoactivable A3 side chain (para-azido-Phe (Pap)). The probe cross-links to a C-terminal peptide (residues 703-719 of the receptor A isoform, KTFEDYLHNVVFVPRPS) containing side chains critical for hormone binding (underlined); the corresponding segment of the holoreceptor was shown previously to cross-link to a PapB²⁵-insulin analog. Because Pap is larger than Val and so may protrude beyond the A3-associated crevice, we investigated analogs containing A3 substitutions comparable in size to Val as follows: Thr, allo-Thr, and α-aminobutyric acid (Aba). Substitutions were introduced within an engineered monomer. Whereas previous studies of smaller substitutions (GlyA³ and SerA³) encountered nonlocal conformational perturbations, NMR structures of the present analogs are similar to wild-type insulin; the variant side chains are accommodated within a native-like crevice with minimal distortion. Receptor binding activities of AbaA³ and allo-ThrA³ analogs are reduced at least 10-fold; the activity of ThrA³-DKP-insulin is reduced 5-fold. The hormone-receptor interface is presumably destabilized either by a packing defect (AbaA³) or by altered polarity (allo-ThrA³ and ThrA³). Our results provide evidence that ValA³, a site of mutation causing diabetes mellitus, contacts the insert domain-derived tail of the α-subunit in a hormone-receptor complex.
Binding of insulin to the insulin receptor plays a central role in the hormonal control of metabolism. Here, we investigate possible contact sites between the receptor and the conserved non-polar ...surface of the B-chain. Evidence is presented that two contiguous sites in an alpha-helix, Val(B12) and Tyr(B16), contact the receptor. Chemical synthesis is exploited to obtain non-standard substitutions in an engineered monomer (DKP-insulin). Substitution of Tyr(B16) by an isosteric photo-activatable derivative (para-azido-phenylalanine) enables efficient cross-linking to the receptor. Such cross-linking is specific and maps to the L1 beta-helix of the alpha-subunit. Because substitution of Val(B12) by larger side-chains markedly impairs receptor binding, cross-linking studies at B12 were not undertaken. Structure-function relationships are instead probed by side-chains of similar or smaller volume: respective substitution of Val(B12) by alanine, threonine, and alpha-aminobutyric acid leads to activities of 1(+/-0.1)%, 13(+/-6)%, and 14(+/-5)% (relative to DKP-insulin) without disproportionate changes in negative cooperativity. NMR structures are essentially identical with native insulin. The absence of transmitted structural changes suggests that the low activities of B12 analogues reflect local perturbation of a "high-affinity" hormone-receptor contact. By contrast, because position B16 tolerates alanine substitution (relative activity 34(+/-10)%), the contribution of this neighboring interaction is smaller. Together, our results support a model in which the B-chain alpha-helix, functioning as an essential recognition element, docks against the L1 beta-helix of the insulin receptor.
T cell sensitization to two myelin components, myelin basic protein (MBP) and myelin proteolipid protein (PLP), may be important to the pathogenesis of multiple sclerosis (MS). Using the limiting ...dilution assay, we demonstrated that the blood of MS patients had an increased frequency of MBP-reactive T cells compared with normal subjects and patients with other neurological diseases (OND) and rheumatoid arthritis. There was no difference in T cell frequency to a synthetic peptide, PLP139-151, or Herpes simplex virus. Within cerebrospinal fluid (CSF), 37% of IL-2/IL-4-reactive T cell isolates from MS patients responded either to MBP or PLP139-151 while only 5% of similar isolates from OND patients responded to these myelin antigens. The mean relative frequency of MBP-reactive T cells within CSF from MS patients was significantly higher than that of OND patients (22 x 10(-5) cells versus 1 x 10(-5) cells) and was similar to that of MBP reactive T cells within the central nervous system of rats with experimental autoimmune encephalomyelitis. These results lend new support to the hypothesis that myelin-reactive T cells mediate disease in MS.
The A and B chains of insulin combine to form native disulfide bridges without detectable isomers. The fidelity of chain combination thus recapitulates the folding of proinsulin, a precursor protein ...in which the two chains are tethered by a disordered connecting peptide. We have recently shown that chain combination is blocked by seemingly conservative substitutions in the C-terminal alpha-helix of the A chain. Such analogs, once formed, nevertheless retain high biological activity. By contrast, we demonstrate here that chain combination is robust to non-conservative substitutions in the N-terminal alpha-helix. Introduction of multiple glycine substitutions into the N-terminal segment of the A chain (residues A1-A5) yields analogs that are less stable than native insulin and essentially without biological activity. (1)H NMR studies of a representative analog lacking invariant side chains Ile(A2) and Val(A3) (A chain sequence GGGEQCCTSICSLYQLENYCN; substitutions are italicized and cysteines are underlined) demonstrate local unfolding of the A1-A5 segment in an otherwise native-like structure. That this and related partial folds retain efficient disulfide pairing suggests that the native N-terminal alpha-helix does not participate in the transition state of the reaction. Implications for the hierarchical folding mechanisms of proinsulin and insulin-like growth factors are discussed.
Proteins evolve in a fitness landscape encompassing a complex network of biological constraints. Because of the interrelation of folding, function, and regulation, the ground-state structure of a ...protein may be inactive. A model is provided by insulin, a vertebrate hormone central to the control of metabolism. Whereas native assembly mediates storage within pancreatic β-cells, the active conformation of insulin and its mode of receptor binding remain elusive. Here, functional surfaces of insulin were probed by photocross-linking of an extensive set of azido derivatives constructed by chemical synthesis. Contacts are circumferential, suggesting that insulin is encaged within its receptor. Mapping of photoproducts to the hormone-binding domains of the insulin receptor demonstrated alternating contacts by the B-chain β-strand (residues B24-B28). Whereas even-numbered probes (at positions B24 and B26) contact the N-terminal L1 domain of the α-subunit, odd-numbered probes (at positions B25 and B27) contact its C-terminal insert domain. This alternation corresponds to the canonical structure of aβ-strand (wherein successive residues project in opposite directions) and so suggests that the B-chain inserts between receptor domains. Detachment of a receptor-binding arm enables photo engagement of surfaces otherwise hidden in the free hormone. The arm and associated surfaces contain sites also required for nascent folding and self-assembly of storage hexamers. The marked compression of structural information within a short polypeptide sequence rationalizes the diversity of diabetes-associated mutations in the insulin gene. Our studies demonstrate that photoscanning mutagenesis can decode the active conformation of a protein and so illuminate cryptic constraints underlying its evolution.
The contribution of the insulin A-chain to receptor binding is investigated by photo-cross-linking and nonstandard mutagenesis. Studies focus on the role of ValA3, which projects within a crevice ...between the A- and B-chains. Engineered receptor α-subunits containing specific protease sites (“midi-receptors”) are employed to map the site of photo-cross-linking by an analog containing a photoactivable A3 side chain (para-azido-Phe (Pap)). The probe cross-links to a C-terminal peptide (residues 703-719 of the receptor A isoform, KTFEDYLHNVVFVPRPS) containing side chains critical for hormone binding (underlined); the corresponding segment of the holoreceptor was shown previously to cross-link to a PapB25-insulin analog. Because Pap is larger than Val and so may protrude beyond the A3-associated crevice, we investigated analogs containing A3 substitutions comparable in size to Val as follows: Thr, allo-Thr, and α-aminobutyric acid (Aba). Substitutions were introduced within an engineered monomer. Whereas previous studies of smaller substitutions (GlyA3 and SerA3) encountered nonlocal conformational perturbations, NMR structures of the present analogs are similar to wild-type insulin; the variant side chains are accommodated within a native-like crevice with minimal distortion. Receptor binding activities of AbaA3 and allo-ThrA3 analogs are reduced at least 10-fold; the activity of ThrA3-DKP-insulin is reduced 5-fold. The hormone-receptor interface is presumably destabilized either by a packing defect (AbaA3) or by altered polarity (allo-ThrA3 and ThrA3). Our results provide evidence that ValA3, a site of mutation causing diabetes mellitus, contacts the insert domain-derived tail of the α-subunit in a hormone-receptor complex.
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