The inhibitor-of-apoptosis proteins (IAPs) regulate programmed cell death by inhibiting members of the caspase family of enzymes. Recently, a mammalian protein called Smac (also named DIABLO) was identified that binds to the IAPs and promotes caspase activation. Although undefined in the X-ray structure, the amino-terminal residues of Smac are critical for its function. To understand the structural basis for molecular recognition between Smac and the IAPs, we determined the solution structure of the BIR3 domain of X-linked IAP (XIAP) complexed with a functionally active nine-residue peptide derived from the N terminus of Smac. The peptide binds across the third beta-strand of the BIR3 domain in an extended conformation with only the first four residues contacting the protein. The complex is stabilized by four intermolecular hydrogen bonds, an electrostatic interaction involving the N terminus of the peptide, and several hydrophobic interactions. This structural information, along with the binding data from BIR3 and Smac peptide mutants reported here, should aid in the design of small molecules that may be used for the treatment of cancers that overexpress IAPs.
The de novo design of peptides and proteins has recently emerged as an approach for investigating protein structure and function. Designed, helical peptides provide model systems for dissecting and quantifying the multiple interactions that stabilize secondary structure formation. De novo design is also useful for exploring the features that specify the stoichiometry and stability of alpha-helical coiled coils and for defining the requirements for folding into structures that resemble native, functional proteins. The design process often occurs in a series of discrete steps. Such steps reflect the hierarchy of forces required for stabilizing tertiary structures, beginning with hydrophobic forces and adding more specific interactions as required to achieve a unique, functional protein.
Inhibitor of apoptosis (IAP) proteins are overexpressed in many cancers and have been implicated in tumor growth, pathogenesis, and resistance to chemo- or radiotherapy. On the basis of the NMR structure of a SMAC peptide complexed with the BIR3 domain of X-linked IAP (XIAP), a novel series of XIAP antagonists was discovered. The most potent compounds in this series bind to the baculovirus IAP repeat 3 (BIR3) domain of XIAP with single-digit nanomolar affinity and promote cell death in several human cancer cell lines. In a MDA-MB-231 breast cancer mouse xenograft model, these XIAP antagonists inhibited the growth of tumors. Close structural analogues that showed only weak binding to the XIAP-BIR3 domain were inactive in the cellular assays and showed only marginal in vivo activity. Our results are consistent with a mechanism in which ligands for the BIR3 domain of XIAP induce apoptosis by freeing up caspases. The present study validates the BIR3 domain of XIAP as a target and supports the use of small molecule XIAP antagonists as a potential therapy for cancers that overexpress XIAP.
An understanding of the forces that contribute to stability is pivotal in solving the protein-folding problem. Classical theory suggests that disulfide bonds stabilize proteins by reducing the entropy of the denatured state. More recent theories have attempted to expand this idea, suggesting that in addition to configurational entropic effects, enthalpic and native-state effects occur and cannot be neglected. Experimental thermodynamic evidence is examined from two sources: (1) the disruption of naturally occurring disulfides, and (2) the insertion of novel disulfides. The data confirm that enthalpic and native-state effects are often significant. The experimental changes in free energy are compared to those predicted by different theories. The differences between theory and experiment are large near 300 K and do not lend support to any of the current theories regarding the stabilization of proteins by disulfide bonds. This observation is a result of not only deficiencies in the theoretical models but also from difficulties in determining the effects of disulfide bonds on protein stability against the backdrop of numerous subtle stabilizing factors (in both the native and denatured states), which they may also affect. Keywords: disulfide bonds; protein stability; thermodynamicsThe determination of the forces that govern protein stability is of fundamental importance for our ability to understand and control the interactions of complex biological molecules. It has been known since the 1960s that the primary structure of a protein dictates its threedimensional fold (see Anfinsen, 1973), yet a comprehensive understanding of the factors that impart thermodynamic stability to proteins is elusive. This is because the tertiary folds of native proteins are defined by a large Reprint requests to: Stephen F. Betz, The DuPont Merck Pharmaceutical Company, Wilmington, Delaware 19880-0328.Abbreviations: BPTI, bovine pancreatic trypsin inhibitor; C,, the concentration of GdmCl at which half the protein is denatured; D-state, the denatured state of a protein; GdmCI, guanidinium chloride; hew, hen egg white; mden, the slope of the line of -AGd versus denaturant concentration; N-state, the native state of a protein; RNase, ribonuclease; Tm, temperature at which half the protein is denatured; t l I z , time required for enzymatic activity to decrease 50%; AC,, the difference in heat capacity between the denatured and native states; AGd, AG for protein denaturation; AGd,HZO, AGd determined from chemical denaturation; AGd, T, AGd determined from thermal denaturation; A H , , , the change in conformational enthalpy between the native and denatured states; N H , , AH for protein denaturation; AH&,,, the change in solvational enthalpy between the native and denatured states; AH,,,, AH for protein denaturation at T,,,; AS,,,, the change in conformational entropy between the native and denatured states; AS,, AS for protein denaturation; Ashyd, the change in solvational entropy between the native and denatured states.
Common structural motifs, such as the cupin domains, are found in enzymes performing different biochemical functions while retaining a similar active site configuration and structural scaffold. The soil bacterium Bacillus subtilis has 20 cupin genes (0.5% of the total genome) with up to 14% of its genes in the form of doublets, thus making it an attractive system for studying the effects of gene duplication. There are four bicupins in B. subtilis encoded by the genes yvrK, yoaN, yxaG, and ywfC. The gene products of yvrK and yoaN function as oxalate decarboxylases with a manganese ion at the active site(s), whereas YwfC is a bacitracin synthetase. Here we present the crystal structure of YxaG, a novel iron-containing quercetin 2,3-dioxygenase with one active site in each cupin domain. Yxag is a dimer, both in solution and in the crystal. The crystal structure shows that the coordination geometry of the Fe ion is different in the two active sites of YxaG. Replacement of the iron at the active site with other metal ions suggests modulation of enzymatic activity in accordance with the Irving-Williams observation on the stability of metal ion complexes. This observation, along with a comparison with the crystal structure of YvrK determined recently, has allowed for a detailed structure-function analysis of the active site, providing clues to the diversification of function in the bicupin family of proteins.
The de novo design of a protein that mimics the properties of natural proteins is an important goal.',2 Although some progress has been made in the design of coiled coils,3 including a de novo designed peptide with native-like behavior,3b all globular proteins designed to date appear to show some of the features of molten globule^.^ Here, we describe a peptide that self-assembles into a protein with native-like physical properties. This was achieved by a strategy which included not only interactions that stabilized the desired three-dimensional structure but also interactions that destabilized potential altemative folds.Several designed four-helix bundles have been prepared with a strict heptad repeat of HBa-HPb-HPc-HBd-HBe-HPtHP, (HB and HP are hydrophobic and hydrophilic amino acids, respect i~e l y ) .~.~ These include tetrameric peptides (designated al peptides), dimeric helix-turn-helix units (az), and single chain proteins (a). The a 2 dimers behave very similarly to the full length Q proteins, hence we have concentrated initial studies on the a 2 peptides. The first design, a2B, contained Leu as its sole hydrophobic r e~i d u e ,~~,~ but its core was unusually flexible compared to native proteins. We therefore replaced many of
The de novo design of peptides and proteins has emerged as an attractive approach for investigating protein structure and function. Here, the design, synthesis, and characterization of a new series of alpha-helical peptides intended to form antiparallel four-stranded coiled coils is described. Computer models were generated without the use of extant protein structures and were used to refine the sequence. The peptides are of the general formula Ncap-(XaZbZcLdZeZfZg)3-Ccap, where X is either Ala, Val, Thr, or Leu, and Ncap and Ccap are sequences designed to satisfy the helices unpaired amide nitrogens and carbonyl oxygens, respectively. The hydrophobic residues (at positions a and d) were chosen so that geometric packing of large and small hydrophobes would favor an antiparallel arrangement. Special attention was also given to residues at the helix--helix interfaces. These residues were chosen to balance potential attractive and repulsive electrostatic forces so that the desired topology was favored while other possible folds were destabilized. Two of the four peptides associate under neutral conditions into the desired tetramers. One of the complexes (a = Val) behaves like a native-like protein as judged by NMR, thermodynamics, and apolar dye (ANS) binding. The other tetrameric complex (a = Leu) exhibits broader NMR resonances, diminished values of delta H and delta Cp, and tight binding of the hydrophobic dye ANS, similar to early designed proteins. These results reinforce the importance of optimizing van der Waals packing interactions in protein design but demonstrate that hydrophobic packing must be balanced with hydrogen-bonding and electrostatic interactions to produce novel native-like proteins.
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