Stabilizing and destabilizing clusters in the hydrophobic core of long two-stranded alpha-helical coiled-coils

Detailed sequence analyses of the hydrophobic core residues of two long two-stranded alpha-helical coiled-coils that differ dramatically in sequence, function, and length were performed (tropomyosin of 284 residues and the coiled-coil domain of the myosin rod of 1086 residues). Three types of regions were present in the hydrophobic core of both proteins: stabilizing clusters and destabilizing clusters, defined as three or more consecutive core residues of either stabilizing (Leu, Ile, Val, Met, Phe, and Tyr) or destabilizing (Gly, Ala, Cys, Ser, Thr, Asn, Gln, Asp, Glu, His, Arg, Lys, and Trp) residues, and intervening regions that consist of both stabilizing and destabilizing residues in the hydrophobic core but no clusters. Subsequently, we designed a series of two-stranded coiled-coils to determine what defines a destabilizing cluster and varied the length of the destabilizing cluster from 3 to 7 residues to determine the length effect of the destabilizing cluster on protein stability. The results showed a dramatic destabilization, caused by a single Leu to Ala substitution, on formation of a 3-residue destabilizing cluster (DeltaT(m) of 17-21 degrees C) regardless of the stability of the coiled-coil. Any further substitution of Leu to Ala that increased the size of the destabilizing cluster to 5 or 7 hydrophobic core residues in length had little effect on stability (DeltaT(m) of 1.4-2.8 degrees C). These results suggested that the contribution of Leu to protein stability is context-dependent on whether the hydrophobe is in a stabilizing cluster or its proximity to neighboring destabilizing and stabilizing clusters.     

Inter- and intrasubunit interactions during the formation of RNA polymerase assembly intermediate

We used yeast two-hybrid and in vitro co-immobilization assays to study the interaction between the Escherichia coli RNA polymerase (RNAP) alpha and beta subunits during the formation of alpha(2)beta, a physiological RNAP assembly intermediate. We show that a 430-amino acid-long fragment containing beta conserved segments F, G, H, and a short part of segment I forms a minimal domain capable of specific interaction with alpha. The alpha-interacting domain is held together by protein-protein interactions between beta segments F and I. Residues in catalytically important beta segments H and I directly participate in alpha binding; substitutions of strictly conserved segment H Asp(1084) and segment I Gly(1215) abolish alpha(2)beta formation in vitro and are lethal in vivo. The importance of these beta amino acids in alpha binding is fully supported by the structural model of the Thermus aquaticus RNAP core enzyme. We also demonstrate that determinants of RNAP assembly are conserved, and that a homologue of beta Asp(1084) in A135, the beta-like subunit of yeast RNAP I, is responsible for interaction with AC40, the largest alpha-like subunit. However, the A135-AC40 interaction is weak compared with the E. coli alpha-beta interaction, and A135 mutation that abolishes the interaction is phenotypically silent. The results suggest that in eukaryotes additional RNAP subunits orchestrate the enzyme assembly by stabilizing weak, but specific interactions of core subunits.     

The crystal structure of mouse Nup35 reveals atypical RNP motifs and novel homodimerization of the RRM domain

The nuclear pore complex mediates the transport of macromolecules across the nuclear envelope (NE). The vertebrate nuclear pore protein Nup35, the ortholog of Saccharomyces cerevisiae Nup53p, is suggested to interact with the NE membrane and to be required for nuclear morphology. The highly conserved region between vertebrate Nup35 and yeast Nup53p is predicted to contain an RNA-recognition motif (RRM) domain. Due to its low level of sequence homology with other RRM domains, the RNP1 and RNP2 motifs have not been identified in its primary structure. In the present study, we solved the crystal structure of the RRM domain of mouse Nup35 at 2.7 A resolution. The Nup35 RRM domain monomer adopts the characteristic betaalphabetabetaalphabeta topology, as in other reported RRM domains. The structure allowed us to locate the atypical RNP1 and RNP2 motifs. Among the RNP motif residues, those on the beta-sheet surface are different from those of the canonical RRM domains, while those buried in the hydrophobic core are highly conserved. The RRM domain forms a homodimer in the crystal, in accordance with analytical ultracentrifugation experiments. The beta-sheet surface of the RRM domain, with its atypical RNP motifs, contributes to homodimerization mainly by hydrophobic interactions: the side-chain of Met236 in the beta4 strand of one Nup35 molecule is sandwiched by the aromatic side-chains of Phe178 in the beta1 strand and Trp209 in the beta3 strand of the other Nup35 molecule in the dimer. This structure reveals a new homodimerization mode of the RRM domain.     

Core side-chain packing and backbone conformation in Lpp-56 coiled-coil mutants

Native proteins exhibit precise geometric packing of atoms in their hydrophobic interiors. Nonetheless, controversy remains about the role of core side-chain packing in specifying and stabilizing the folded structures of proteins. Here we investigate the role of core packing in determining the conformation and stability of the Lpp-56 trimerization domain. The X-ray crystal structures of Lpp-56 mutants with alanine substitutions at two and four interior core positions reveal trimeric coiled coils in which the twist of individual helices and the helix-helix spacing vary significantly to achieve the most favored superhelical packing arrangement. Introduction of each alanine "layer" into the hydrophobic core destabilizes the superhelix by 1.4 kcal mol(-1). Although the methyl groups of the alanine residues pack at their optimum van der Waals contacts in the coiled-coil trimer, they provide a smaller component of hydrophobic interactions than bulky hydrophobic side-chains to the thermodynamic stability. Thus, specific side-chain packing in the hydrophobic core of coiled coils are important determinants of protein main-chain conformation and stability.     

The role of a hydrogen bonding network in the transmembrane beta-barrel OMPLA

The hydrogen bonding of polar side-chains has emerged as an important theme for membrane protein interactions. The crystal structure of the dimeric state of the transmembrane beta-barrel protein outer membrane phospholipase A (OMPLA) revealed an intermolecular hydrogen bond mediated by a highly conserved glutamine side-chain (Q94). It has been shown that the introduction of a polar residue can drive the association of model helices, and by extension it was presumed that the glutamine hydrogen bond played a key role in stabilizing the OMPLA dimer. However, a thermodynamic investigation using sedimentation equilibrium ultracentrifugation in detergent micelles reveals that the hydrogen bond plays only a very modest role in stabilizing the dimer. The Q94 side-chain is hydrogen bonded intramolecularly to residues Y92 and S96, but amino acid substitutions at these positions suggest these intramolecular interactions are not responsible for attenuating the strength of the intermolecular Q94 hydrogen bond. Other substitutions suggested that hydration of the local environment around Q94 may be responsible for the modest strength of the hydrogen bond. Heat inactivation experiments with the variants suggest that the Y92-Q94-S96 network may instead be important for thermal stability of the monomer. These results highlight the context dependence and broad range of interactions that can be mediated by polar residues in membrane proteins.     

Structural basis for activation of Swi2/Snf2 ATPase RapA by RNA polymerase

RapA is a bacterial RNA polymerase (RNAP)-associated Swi2/Snf2 ATPase that stimulates RNAP recycling. The ATPase activity of RapA is autoinhibited by its N-terminal domain (NTD) but activated with RNAP bound. Here, we report a 3.4-Å cryo-EM structure of Escherichia coli RapA–RNAP elongation complex, in which the ATPase active site of RapA is structurally remodeled. In this process, the NTD of RapA is wedged open by RNAP β'' zinc-binding domain (ZBD). In addition, RNAP β flap tip helix (FTH) forms extensive hydrophobic interactions with RapA ATPase core domains. Functional assay demonstrates that removing the ZBD or FTH of RNAP significantly impairs its ability to activate the ATPase activity of RapA. Our results provide the structural basis of RapA ATPase activation by RNAP, through the active site remodeling driven by the ZBD-buttressed large-scale opening of NTD and the direct interactions between FTH and ATPase core domains.     

Importance of homodimerization for the in vivo function of yeast RNA triphosphatase

Saccharomyces cerevisiae RNA triphosphatase Cet1 is an essential component of the yeast mRNA capping apparatus. The active site of Cet1 resides within a topologically closed hydrophilic beta-barrel (the triphosphate tunnel) that is supported by a globular hydrophobic core. The homodimeric quaternary structure of Cet1 is formed by a network of contacts between the partner protomers. By studying the effects of alanine-cluster mutations, we highlight the contributions of two separate facets of the crystallographic dimer interface to Cet1 function in vivo. One essential facet of the interface entails hydrophobic cross-dimer interactions of Cys(330) and Val(331) and a cross-dimer hydrogen bond of Asp(280) with the backbone amide of Gln(329). The second functionally relevant dimer interface involves hydrophobic side-chain interactions of Phe(272) and Leu(273). Ala-cluster mutations involving these residues elicited lethal or severe temperature-sensitive phenotypes that were suppressed completely by fusion of the mutated triphosphatases to the guanylyltransferase domain of mammalian capping enzyme. The recombinant D279A-D280A and F272A-L273A proteins retained phosphohydrolase activity but sedimented as monomers. These results indicate that a disruption of the dimer interface is uniquely deleterious when the yeast RNA triphosphatase must function in concert with the endogenous yeast guanylyltransferase. We also identify key residue pairs in the hydrophobic core of the Cet1 protomer that support the active site tunnel and stabilize the triphosphatase in vivo.     

Solid-State NMR Evidence for Inequivalent GvpA Subunits in Gas Vesicles

Gas vesicles are organelles that provide buoyancy to the aquatic microorganisms that harbor them. The gas vesicle shell consists almost exclusively of the hydrophobic 70-residue gas vesicle protein A, arranged in an ordered array. Solid-state NMR spectra of intact collapsed gas vesicles from the cyanobacterium Anabaena flos-aquae show duplication of certain gas vesicle protein A resonances, indicating that specific sites experience at least two different local environments. Interpretation of these results in terms of an asymmetric dimer repeat unit can reconcile otherwise conflicting features of the primary, secondary, tertiary, and quaternary structures of the gas vesicle protein. In particular, the asymmetric dimer can explain how the hydrogen bonds in the β-sheet portion of the molecule can be oriented optimally for strength while promoting stabilizing aromatic and electrostatic side-chain interactions among highly conserved residues and creating a large hydrophobic surface suitable for preventing water condensation inside the vesicle.     

The role of aromatic residues in the hydrophobic core of the villin headpiece subdomain

Small autonomously folding proteins are of interest as model systems to study protein folding, as the same molecule can be used for both experimental and computational approaches. The question remains as to how well these minimized peptide model systems represent larger native proteins. For example, is the core of a minimized protein tolerant to mutation like larger proteins are? Also, do minimized proteins use special strategies for specifying and stabilizing their folded structure? Here we examine these questions in the 35‐residue autonomously folding villin headpiece subdomain (VHP subdomain). Specifically, we focus on a cluster of three conserved phenylalanine (F) residues F47, F51, and F58, that form most of the hydrophobic core. These three residues are oriented such that they may provide stabilizing aromatic–aromatic interactions that could be critical for specifying the fold. Circular dichroism and 1D‐NMR spectroscopy show that point mutations that individually replace any of these three residues with leucine were destabilized, but retained the native VHP subdomain fold. In pair‐wise replacements, the double mutant that retains F58 can adopt the native fold, while the two double mutants that lack F58 cannot. The folding of the double mutant that retains F58 demonstrates that aromatic–aromatic interactions within the aromatic cluster are not essential for specifying the VHP subdomain fold. The ability of the VHP subdomain to tolerate mutations within its hydrophobic core indicates that the information specifying the three dimensional structure is distributed throughout the sequence, as observed in larger proteins. Thus, the VHP subdomain is a legitimate model for larger, native proteins.     

Crystallographic studies of human MitoNEET

MitoNEET was identified as an outer mitochondrial membrane protein that can potentially bind the anti-diabetes drug pioglitazone. The crystal structure of the cytoplasmic mitoNEET (residues 33-108) is determined in this study. The structure presents a novel protein fold and contains a [2Fe-2S] cluster-binding domain. The [2Fe-2S] cluster is coordinated to the protein by Cys-72, Cys-74, Cys-83, and His-87 residues. This coordination is also novel compared with the traditional [2Fe-2S] cluster coordinated by four cysteines or two cysteines and two histidines. The cytoplasmic mitoNEET forms homodimers in solution and in crystal. The dimerization is mainly mediated by hydrophobic interactions as well as hydrogen bonds coordinated by two water molecules binding at the interface. His-87 residue, which plays an important role in the coordination of the [2Fe-2S] cluster, is exposed to the solvent on the dimer surface. It is proposed that mitoNEET dimer may interact with other proteins via the surface residues in close proximity to the [2Fe-2S] cluster.     

Distinct functions of regions 1.1 and 1.2 of RNA polymerase σ subunits from Escherichia coli and Thermus aquaticus in transcription initiation

RNA polymerase (RNAP) from thermophilic Thermus aquaticus is characterized by higher temperature of promoter opening, lower promoter complex stability, and higher promoter escape efficiency than RNAP from mesophilic Escherichia coli. We demonstrate that these differences are in part explained by differences in the structures of the N-terminal regions 1.1 and 1.2 of the E. coli σ(70) and T. aquaticus σ(A) subunits. In particular, region 1.1 and, to a lesser extent, region 1.2 of the E. coli σ(70) subunit determine higher promoter complex stability of E. coli RNAP. On the other hand, nonconserved amino acid substitutions in region 1.2, but not region 1.1, contribute to the differences in promoter opening between E. coli and T. aquaticus RNAPs, likely through affecting the σ subunit contacts with DNA nucleotides downstream of the -10 element. At the same time, substitutions in σ regions 1.1 and 1.2 do not affect promoter escape by E. coli and T. aquaticus RNAPs. Thus, evolutionary substitutions in various regions of the σ subunit modulate different steps of the open promoter complex formation pathway, with regions 1.1 and 1.2 affecting promoter complex stability and region 1.2 involved in DNA melting during initiation.     

Crystal structure of Escherichia coli SufA involved in biosynthesis of iron-sulfur clusters: implications for a functional dimer

IscA and SufA are paralogous proteins that play crucial roles in the biosynthesis of Fe-S clusters, perhaps through a mechanism involving transient Fe-S cluster formation. We have determined the crystal structure of E. coli SufA at 2.7A resolution. SufA exists as a homodimer, in contrast to the tetrameric organization of IscA. Furthermore, a C-terminal segment containing two essential cysteine residues (Cys-Gly-Cys), which is disordered in the IscA structure, is clearly visible in one molecule (the alpha1 subunit) of the SufA homodimer. Although this segment is disordered in the other molecule (the alpha2 subunit), computer modeling of this segment based on the well-defined conformation of alpha1 subunit suggests that the four cysteine residues (Cys114 and Cys116 in each subunit) in the Cys-Gly-Cys motif are positioned in close proximity at the dimer interface. The arrangement of these cysteines together with the nearby Glu118 in SufA dimer may allow coordination of an Fe-S cluster and/or an Fe atom.     

Structural Analysis of Sindbis Virus Capsid Mutants Involving Assembly and Catalysis

Sindbis virus core protein (SCP) has been isolated from virus and crystallized. The X-ray crystallographic structure showed that the amino-terminal 113 residues appeared to be either disordered or truncated during crystallization and that the carboxy-terminal residues 114 to 264 had a chymotrypsin-like structure. The carboxy-terminal residues 106 to 264 and 106 to 266 of SCP have now been expressed inEscherichia coli. Most crystal forms of the truncated proteins were isomorphous with those of the virally extracted protein. There are only small structural differences between the truncated recombinant protein and the ordered part of the wild-type virus-extracted protein. Hence,E. coli-expressed SCP can be used to study proteolytic properties and the contribution of SCP to nucleocapsid assembly, interaction with the E2 glycoprotein and interaction with RNA.The same dimer that was found in two different crystal forms of the virus-extracted SCP was present also in some of the crystals of the truncated recombinant protein. The monomer – monomer interface is maintained by two pairs of hydrogen bonds and by hydrophobic interactions. Removal of the hydrogen bonds by single substitutions did not prevent dimer formation. However, a mutation that reduced the hydrophobic contacts did inhibit dimer formation.The wild-type truncated SCP is active inE. coli, as evidenced by proteolytic processing of a series of progressively longer precursors that extend beyond residue 264. Unlike the virus-extracted capsid protein, theE. coli-expressed SCP described here is terminated following the carboxy-terminal residue and, therefore, does not require autocatalysis. Nevertheless, theE. coli-expressed protein folds with the carboxy-terminal tryptophan residue in the specificity pocket. Two crystallographically independent molecules of SCP(106 to 266), which had two additional downstream residues and had the essential S215 mutated to alanine, showed two distinct modes of binding the uncleaved carboxy-terminal residues. These may represent successive steps of binding substrate prior to catalytic cleavage.Refinement of the various crystal structures of SCP showed that the amino-terminal arm from residues 107 to 113 was not disordered, but is associated with neighboring molecules. Residues 108 to 111 bind into a hydrophobic pocket composed primarily of Y180, W247 and F166. It had been shown that the double mutant (Y180S; E183G), with the Y180S substitution in this pocket, produced a large number of non-infectious virions, possibly because of modification in the interaction of the glycoprotein spikes with core proteins. The crystal structure of this double mutant showed that there was a large positional change in the side-chain of W247, which moved into the space created by the replacement of Y180 with serine. These conformational changes may alter the stability of the virion and, thus, regulate its functional requirements during cell entry.     

The SufE sulfur-acceptor protein contains a conserved core structure that mediates interdomain interactions in a variety of redox protein complexes

The isc and suf operons in Escherichia coli represent alternative genetic systems optimized to mediate the essential metabolic process of iron-sulfur cluster (Fe-S) assembly under basal or oxidative-stress conditions, respectively. Some of the proteins in these two operons share strong sequence homology, e.g. the cysteine desulfurases IscS and SufS, and presumably play the same role in the oxygen-sensitive assembly process. However, other proteins in these operons share no significant homology and occur in a mutually exclusive manner in Fe-S assembly operons in other organisms (e.g. IscU and SufE). These latter proteins presumably play distinct roles adapted to the different assembly mechanisms used by the two systems. IscU has three invariant cysteine residues that function as a template for Fe-S assembly while accepting a sulfur atom from IscS. SufE, in contrast, does not function as an Fe-S assembly template but has been suggested to function as a shuttle protein that uses a persulfide linkage to a single invariant cysteine residue to transfer a sulfur atom from SufS to an alternative Fe-S assembly template. Here, we present and analyze the 2.0A crystal structure of E.coli SufE. The structure shows that the persulfide-forming cysteine occurs at the tip of a loop with elevated B-factors, where its side-chain is buried from solvent exposure in a hydrophobic cavity located beneath a highly conserved surface. Despite the lack of sequence homology, the core of SufE shows strong structural similarity to IscU, and the sulfur-acceptor site in SufE coincides with the location of the cysteine residues mediating Fe-S cluster assembly in IscU. Thus, a conserved core structure is implicated in mediating the interactions of both SufE and IscU with the mutually homologous cysteine desulfurase enzymes present in their respective operons. A similar core structure is observed in a domain found in a variety of Fe-S cluster containing flavoenzymes including xanthine dehydrogenase, where it also mediates interdomain interactions. Therefore, the core fold of SufE/IscU has been adapted to mediate interdomain interactions in diverse redox protein systems in the course of evolution.