Guy Dodson

Insulin: The Structure in the Crystal and its Reflection in Chemistry and Biology by

Publisher Summary This chapter reviews the physical, chemical, and biological properties of insulin in the light of the atomic arrangement found in insulin crystals. It also describes the relation of the three-dimensional arrangement of the atoms in the molecule of 2-zinc insulin crystal to the solution properties of insulin (particularly its states of aggregation), to the chemical reaction and chemical modification of the molecule, and to its primary biological activity. Normally the insulin crystals contain two zinc ions to every six molecules of insulin—a hexamer. The slow solution of the crystals provides a method of delaying the action of insulin that closely parallels the methods adopted in the pancreas itself for the storage and release of insulin. Within many β granules, grains can be seen that almost certainly contain zinc insulin hexamers packed in a crystalline array, and in experimental animals diabetes has been induced by chelating agents, such as EDTA, perhaps simply by interfering with normal insulin storage. It, therefore, seems plausible that ready crystallization of insulin in the presence of zinc is a reflection of the storage processes in the β cell.

Catalytic triads and their relatives

Interactions among the residues in the serine protease Asp-His-Ser catalytic triad, in the special environment of the enzyme-substrate complex, activate the nucleophilic potential of the seryl O gamma. In the subtilisin and trypsin families, the composition and arrangement of the catalytic triad do not vary significantly. However, the mechanisms of action of many other hydrolytic enzymes, which target a wide range of substrates, involve nucleophilic attack by a serine (or threonine) residue. Review of these enzymes shows that the acid-base-ser/thr pattern of catalytic residues is generally conserved, although the individual acids and bases can vary. The variations in sequence and organization illustrate the adaptability shown by proteins in generating catalytic stereochemistry on different main-chain frameworks.

The role of assembly in insulin's biosynthesis

Insulin is synthesised as a single-chain precursor, preproinsulin, that contains an N-terminal signal sequence and a connecting peptide linking the A and B chains of the insulin molecule. Nascent proinsulin is directed into the regulated secretory pathway, converted to insulin and stored as microcrystals. These processes exploit assembly to the zinc-containing hexamer. Structural, chemical and genetic studies, and experiments with transgenic animals and transfected cells are providing new details about the molecular events in insulins biosynthesis.

Structure of the trp RNA-binding attenuation protein, TRAP, bound to RNA

The trp RNA-binding attenuation protein (TRAP) regulates expression of the tryptophan biosynthetic genes of several bacilli by binding single-stranded RNA. The binding sequence is composed of eleven triplet repeats, predominantly GAG, separated by two or three non-conserved nucleotides. Here we present the crystal structure of a complex of TRAP and a 53-base single-stranded RNA containing eleven GAG triplets, revealing that each triplet is accommodated in a binding pocket formed by β-strands. In the complex, the RNA has an extended structure without any base-pairing and binds to the protein mostly by specific protein–base interactions. Eleven binding pockets on the circular TRAP 11-mer form a belt with a diameter of about 80 Å. This simple but elegant mechanism of arresting the RNA segment by encircling it around a protein disk is applicable to both transcription, when TRAP binds the nascent RNA, and to translation, when TRAP binds the same sequence within a non-coding leader region of the messenger RNA.

How insulin engages its primary binding site on the insulin receptor.

Insulin receptor signalling has a central role in mammalian biology, regulating cellular metabolism, growth, division, differentiation and survival. Insulin resistance contributes to the pathogenesis of type 2 diabetes mellitus and the onset of Alzheimer’s disease; aberrant signalling occurs in diverse cancers, exacerbated by cross-talk with the homologous type 1 insulin-like growth factor receptor (IGF1R). Despite more than three decades of investigation, the three-dimensional structure of the insulin–insulin receptor complex has proved elusive, confounded by the complexity of producing the receptor protein. Here we present the first view, to our knowledge, of the interaction of insulin with its primary binding site on the insulin receptor, on the basis of four crystal structures of insulin bound to truncated insulin receptor constructs. The direct interaction of insulin with the first leucine-rich-repeat domain (L1) of insulin receptor is seen to be sparse, the hormone instead engaging the insulin receptor carboxy-terminal α-chain (αCT) segment, which is itself remodelled on the face of L1 upon insulin binding. Contact between insulin and L1 is restricted to insulin B-chain residues. The αCT segment displaces the B-chain C-terminal β-strand away from the hormone core, revealing the mechanism of a long-proposed conformational switch in insulin upon receptor engagement. This mode of hormone–receptor recognition is novel within the broader family of receptor tyrosine kinases. We support these findings by photo-crosslinking data that place the suggested interactions into the context of the holoreceptor and by isothermal titration calorimetry data that dissect the hormone–insulin receptor interface. Together, our findings provide an explanation for a wealth of biochemical data from the insulin receptor and IGF1R systems relevant to the design of therapeutic insulin analogues.

Role of zinc in insulin biosynthesis

SummaryThe behaviour of proinsulin and insulin in the presence of zinc suggests it plays an important role in insulins production in the B-cell for the vast majority of animal species. The postulate that proinsulin forms a zinc containing hexamer soon after its synthesis and that this organization of the molecule is maintained through all the subsequent processes is supported by our observation that the proinsulin hexamer is converted readily into the insulin hexamer. In addition the zinc ions enhance proinsulins solubility and render insulin insoluble. Zinc ions also appear to play an important role in the microcrystalline character of the precipitated insulin granule. There may be advantages in condensing the stored material in this way; it will reduce contact with the surrounding membrane where the converting, and possibly other enzymes, are thought to be located, and it will tend to exclude incompletely converted hexamers.

Insulin at pH 2: Structural analysis of the conditions promoting insulin fibre formation

When insulin solutions are subjected to acid, heat and agitation, the normal pattern of insulin assembly (dimers-->tetramers-->hexamers) is disrupted; the molecule undergoes conformational changes allowing it to follow an alternative aggregation pathway (via a monomeric species) leading to the formation of insoluble amyloid fibres. To investigate the effect of acid pH on the conformation and aggregation state of the protein, the crystal structure of human insulin at pH 2.1 has been determined to 1.6 A resolution. The structure reveals that the native fold is maintained at low pH, and that the molecule is still capable of forming dimers similar to those found in hexameric insulin structures at higher pH. Sulphate ions are incorporated into the molecule and the crystal lattice where they neutralise positive charges on the protein, stabilising its structure and facilitating crystallisation. The sulphate interactions are associated with local deformations in the protein, which may indicate that the structure is more plastic at low pH. Transmission electron microscopy analysis of insulin fibres reveals that the appearance of the fibres is greatly influenced by the type of acid employed. Sulphuric acid produces distinctive highly bunched, truncated fibres, suggesting that the sulphate ions have a sophisticated role to play in fibre formation, rather as they do in the crystal structure. Analytical ultracentrifugation studies show that in the absence of heating, insulin is predominantly dimeric in mineral acids, whereas in acetic acid the equilibrium is shifted towards the monomer. Hence, the effect of acid on the aggregation state of insulin is also complex. These results suggest that acid conditions increase the susceptibility of the molecule to conformational change and dissociation, and enhance the rate of fibrillation by providing a charged environment in which the attractive forces between the protein molecules is increased.

Transmission of conformational change in insulin

Crystal structures of insulin contain molecules that are similar but not identical in conformation. Packed helices move relative to each other, these shifts being accommodated by motions of side-chain atoms arising from small changes in torsion angles. Such low-energy conformational adjustments can accommodate shifts of no more than ∼1.5 Å. This limits the extent to which conformational changes can be dissipated locally, causing their transmission over long distances.

The structural and sequence homology of a family of microbial ribonucleases

Abstract The crystal structures of five microbial ribonucleases have been determined in the last 2 years. They have been compared by the laboratories concerned in a collaborative exercise. Homology in the structure and sequence is present in the C-terminal half of the polypeptide chain, which is folded as a four-stranded, anti-parallel β-sheet containing the catalytic residues which are consistent with the idea of a diverging family of enzymes. The prokaryotic and eukaryotic enzymes are distinguished by a different design around the catalytic histidine. While the tertiary structures (particularly in the N-terminal half of the chain) in the two classes of ribonucleases are also notably different there are also large variations in the folding within the prokaryotic group of enzymes.

Structure of the intact transactivation domain of the human papillomavirus E2 protein.

Papillomaviruses cause warts and proliferative lesions in skin and other epithelia. In a minority of papillomavirus types (‘high risk’, including human papillomaviruses 16, 18, 31, 33, 45 and 56), further transformation of the wart lesions can produce tumours. The papillomavirus E2 protein controls primary transcription and replication of the viral genome. Both activities are governed by a ∼200 amino-acid amino-terminal module (E2NT) which is connected to a DNA-binding carboxy-terminal module by a flexible linker. Here we describe the crystal structure of the complete E2NT module from human papillomavirus 16. The E2NT module forms a dimer both in the crystal and in solution. Amino acids that are necessary for transactivation are located at the dimer interface, indicating that the dimer structure may be important in the interactions of E2NT with viral and cellular transcription factors. We propose that dimer formation may contribute to the stabilization of DNA loops which may serve to relocate distal DNA-binding transcription factors to the site of human papillomavirus transcription initiation.