John Orban

Solution structure of the pro-hormone convertase 1 pro-domain from Mus musculus.

The solution structure of the mouse pro-hormone convertase (PC) 1 pro-domain was determined using heteronuclear NMR spectroscopy and is the first structure to be obtained for any of the domains in the convertase family. The ensemble of NMR-derived structures shows a well-ordered core consisting of a four-stranded antiparallel beta-sheet with two alpha-helices packed against one side of this sheet. Sequence homology suggests that the other eukaryotic PC pro-domains will have the same overall fold and most of the residues forming the hydrophobic core of PC1 are highly conserved within the PC family. However, some of the core residues are predicted by homology to be replaced by polar amino acid residues in other PC pro-domains and this may help to explain their marginal stability. Interestingly, the folding topology observed here is also seen for the pro-domain of bacterial subtilisin despite little or no sequence homology. Both the prokaryotic and eukaryotic structures have hydrophobic residues clustered on the solvent-accessible surface of their beta-sheets although the individual residue types differ. In the bacterial case this region is buried at the binding interface with the catalytic domain and, in the eukaryotic PC family, these surface residues are conserved. We therefore propose that the hydrophobic patch in the PC1 pro-domain is involved in the binding interface with its cognate catalytic domain in a similar manner to that seen for the bacterial system. The PC1 pro-domain structure also reveals potential mechanisms for the acid-induced dissociation of the complex between pro- and catalytic domains.

De novo structure generation using chemical shifts for proteins with high‐sequence identity but different folds

Proteins with high‐sequence identity but very different folds present a special challenge to sequence‐based protein structure prediction methods. In particular, a 56‐residue three‐helical bundle protein (GA95) and an α/β‐fold protein (GB95), which share 95% sequence identity, were targets in the CASP‐8 structure prediction contest. With only 12 out of 300 submitted server‐CASP8 models for GA95 exhibiting the correct fold, this protein proved particularly challenging despite its small size. Here, we demonstrate that the information contained in NMR chemical shifts can readily be exploited by the CS‐Rosetta structure prediction program and yields adequate convergence, even when input chemical shifts are limited to just amide 1HN and 15N or 1HN and 1Hα values.

An artificially evolved albumin binding module facilitates chemical shift epitope mapping of GA domain interactions with phylogenetically diverse albumins

Protein G‐related albumin‐binding (GA) modules occur on the surface of numerous Gram‐positive bacterial pathogens and their presence may promote bacterial growth and virulence in mammalian hosts. We recently used phage display selection to evolve a GA domain, PSD‐1 (phage selected domain‐1), which tightly bound phylogenetically diverse albumins. With respect to PSD‐1s broad albumin binding specificity, it remained unclear how the evolved binding epitope compared to those of naturally occurring GA domains and whether PSD‐1s binding mode was the same for different albumins. We investigate these questions here using chemical shift perturbation measurements of PSD‐1 with rabbit serum albumin (RSA) and human serum albumin (HSA) and put the results in the context of previous work on structure and dynamics of GA domains. Combined, these data provide insights into the requirements for broad binding specificity in GA‐albumin interactions. Moreover, we note that using the phage‐optimized PSD‐1 protein significantly diminishes the effects of exchange broadening at the binding interface between GA modules and albumin, presumably through stabilization of a ligand‐bound conformation. The employment of artificially evolved domains may be generally useful in NMR structural studies of other protein–protein complexes.

Solution structure of the highly acidic protein HI1450 from Haemophilus influenzae, a putative double-stranded DNA mimic.

The solution structure of the acidic protein HI1450 from Haemophilus influenzae has been determined by NMR spectroscopy. HI1450 has homologues in ten other bacterial species including Escherichia coli, Vibrio cholerae, and Yersinia pestis but there are no functional assignments for any members of the family. Thirty‐one of the amino acids in this 107‐residue protein are aspartates or glutamates, contributing to an unusually low pI of 3.72. The secondary structure elements are arranged in an α‐α‐β‐β‐β‐β order with the two alpha helices packed against the same side of an anti‐parallel four‐stranded beta meander. Two large loops, one between β1 and β2 and the other between β2 and β3 bend almost perpendicularly across the β‐strands in opposite directions on the non‐helical side of the β‐sheet to form a conserved hydrophobic cavity. The HI1450 structure has some similarities to the structure of the double‐stranded DNA (dsDNA) mimic uracil DNA glycosylase inhibitor (Ugi) including the distribution of surface charges and the position of the hydrophobic cavity. Based on these similarities, as well as having a comparable molecular surface to dsDNA, we propose that HI1450 may function as a dsDNA mimic in order to inhibit or regulate an as yet unidentified dsDNA binding protein. Proteins 2004;54:000–000.

HU‐α binds to the putative double‐stranded DNA mimic HI1450 from Haemophilus influenzae

Recently, the solution structure of the hypothetical protein HI1450 from Haemophilus influenzae was solved as part of a structure‐based effort to understand function. The distribution of its many negatively charged residues and weak structure and sequence homology to uracil DNA glycosylase inhibitor (Ugi) suggested that HI1450 may act as a double‐stranded DNA (dsDNA) mimic. We present supporting evidence here and show that HI1450 interacts with the dsDNA‐binding protein HU‐α. The interaction between HI1450 and HU‐α from H. influenzae is characterized using calorimetry and NMR spectroscopy. HU‐α binds to HI1450 with a Kd of 3.0 ± 0.2 μM, which is similar in affinity to its interaction with dsDNA. Chemical shift perturbation data indicate that the β1‐strand of HI1450 and neighboring regions are most directly involved in interactions with HU‐α. These results show that HI1450 and its structural homolog, Ugi, use similar parts of their structures to recognize DNA‐binding proteins.

NMR structure of HI0004, a putative essential gene product from Haemophilus influenzae, and comparison with the X-ray structure of an Aquifex aeolicus homolog

The solution structure of the 154‐residue conserved hypothetical protein HI0004 has been determined using multidimensional heteronuclear NMR spectroscopy. HI0004 has sequence homologs in many organisms ranging from bacteria to humans and is believed to be essential in Haemophilus influenzae, although an exact function has yet to be defined. It has a α–β–α sandwich architecture consisting of a central four‐stranded β‐sheet with the α2‐helix packed against one side of the β‐sheet and four α‐helices (α1, α3, α4, α5) on the other side. There is structural homology with the eukaryotic matrix metalloproteases (MMPs), but little sequence similarity except for a conserved region containing three histidines that appears in both the MMPs and throughout the HI0004 family of proteins. The solution structure of HI0004 is compared with the X‐ray structure of an Aquifex aeolicus homolog, AQ_1354, which has 36% sequence identity over 148 residues. Despite this level of sequence homology, significant differences exist between the two structures. These differences are described along with possible functional implications of the structures.

Main chain NMR assignments of subtilisin Sbt70 in its prodomain-bound state

Main chain assignments are described for a 266-residue subtilisin mutant, Sbt70, in its 35xa0kDa complex with an N-terminal prodomain. The assignments provide the basis for understanding how the prodomain assists folding of subtilisin at a residue-specific level.

Solution structure of HI1506, a novel two-domain protein from Haemophilus influenzae.

HI1506 is a 128‐residue hypothetical protein of unknown function from Haemophilus influenzae. It was originally annotated as a shorter 85‐residue protein, but a more detailed sequence analysis conducted in our laboratory revealed that the full‐length protein has an additional 43 residues on the C terminus, corresponding with a region initially ascribed to HI1507. As part of a larger effort to understand the functions of hypothetical proteins from Gram‐negative bacteria, and H. influenzae in particular, we report here the three‐dimensional solution NMR structure for the corrected full‐length HI1506 protein. The structure consists of two well‐defined domains, an α/β 50‐residue N‐domain and a 3‐α 32‐residue C‐domain, separated by an unstructured 30‐residue linker. Both domains have positively charged surface patches and weak structural homology with folds that are associated with RNA binding, suggesting a possible functional role in binding distal nucleic acid sites.