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4 LRP: Leucine Responsive Regulatory Protein

4 LRP: Leucine Responsive Regulatory Protein

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Ü. Pul and R. Wagner

the bacterial gene expression in response to the nutritional quality (Yokoyama

et al. 2006).

The cellular concentration of LRP strongly depends on the availability of nutrients and is inversely proportional to the bacterial growth rate (Landgraf et al. 1996).

DNA microarray analysis has shown that at least 10% of E. coli genes are affected

by LRP; most of these genes are involved in the adaptation of E. coli to nutrient

limitations (Tani et al. 2002).

The oligomerization state of many FFRPs can be regulated through allosteric

effector molecules. In the case of E. coli LRP the amino acid leucine is the allosteric effector. This is a unique feature among the NAP family members for which

otherwise no allosteric effector molecules are known. Through allosteric modulation of the quarternary structure leucine influences the association state of E. coli

LRP, which results in altered DNA binding affinity (Chen and Calvo 2002).The

resulting regulatory effects for individual promoters differ, however. Hence,

among the LRP-regulated genes leucine can either enhance, reduce or have no

effect on binding of LRP (Calvo and Matthews 1994). The association reactions

of LRP are complex and it is assumed that at nanomolar concentrations LRP dimers are the predominant species. Determination of the LRP-DNA stoichiometry

indicated that LRP can bind as a dimer but higher aggregates are frequently found

in DNA complexes (Chen et al. 2005). At micromolar concentrations LRP exists

as octamers and hexadecamers, which in the presence of leucine dissociate preferentially to octamers (Chen et al. 2001; Chen and Calvo 2002). The exact mechanism behind the differential effect of leucine on LRP binding is not fully

understood, however.

The three-dimensional structure of E. coli LRP has been solved by X-ray crystallography (de los Rios and Perona 2007). Figure 8.3a shows the modular structure of an LRP monomer, which is characterized by two structural domains also

reflecting the different functions of LRP. The N-terminal domain contains the

helix-turn-helix DNA binding motif, consisting of three a-helices. According to

mutational analysis the a3-helix of this motif was found to be responsible for

binding to the major groove of the DNA (Platko and Calvo 1993). The C-terminal

domain of LRP contains two a-helices flanking four antiparallel b-strands, forming a ab-sandwich. The resulting babbab-structure constitutes a structural motif

found in many LRP-like regulatory proteins or enzymes involved in amino acid

metabolism and has been termed RAM domain (regulation of amino acid metabolism) (Ettema et al. 2002). Within the RAM domain the b3–b4 loop contains the

binding site for the effector amino acid leucine. The N- and C-terminal domains

are linked by a flexible linker, containing a b-strand (depicted as ‘crossover

b-strand’ in Fig. 8.3a according to de los Rios and Perona (2007)), which upon

dimerization forms a two-stranded antiparallel b-sheet with the corresponding

b-strand of the second monomer at the homodimer centre (Fig. 8.3b). In addition,

dimerization is attributed to hydrophobic interactions between the two b-sheets of

the RAM domains (Chapter 15).

As shown in Fig. 8.3c four LRP dimers associate to form an octamer, arranged

as a disc, in a similar way as has been described for LrpA from Pyrococcus furi-



8  Nucleoid-Associated Proteins: Structural Properties

Fig. 8.3  Crystal structure of E. coli LRP. (PDB ID: 2GQQ; de los Rios and Perona 2007). The

structures of an LRP monomer (a) and dimer (b) are shown. The N-terminal domain contains the

HTH motif, coloured in red. The recognition helix (a3) binds to the major groove of the DNA. The

RAM domain (regulation of amino acid metabolism), is coloured in yellow and contains the binding site for the effector leucine. The crossover b-strand involved in dimerization is coloured in

blue. The a-helices and b-strands are numbered according to the original publication (de los Rios

and Perona 2007). (c) The octameric structure of LRP has been arranged according to the crystallographic data of de los Rios and Perona (2007) by PyMol (DeLano Scientific LLC, South San

Francisco, USA)

ousus (Leonard et al. 2001) and LrpC from Bacillus subtilis (Thaw et al. 2006). The

octameric structure is maintained by hydrophobic interactions of the RAM domains

between individual dimer units. In contrast to the octameric structure of E. coli


Ü. Pul and R. Wagner

LRP, where the interaction of two of the four LRP dimers is interrupted, the octamers of LrpA and LrpC are closed octameric discs. It is possible, however, that this

difference in the quarternary structure is due to the presence of DNA during crystallization of the E. coli LRP (de los Rios and Perona 2007).

Binding of LRP to DNA occurs through contacts with the a3-helix of the HTH

motif (see above) and the DNA major groove. Specificity depends on a consensus

DNA sequence, which had been determined as (T/C)AG(A/C/T)A(A/T)ATT(A/T)

T(A/G/T)CT(G/A) by a SELEX analysis (Cui et al. 1995). For most LRP-regulated

operons multiple LRP binding sites have been mapped, to which binding of LRP

often occurs with high cooperativity. This is in contrast to the occupation of FIS

binding sites, for instance, which are also generally arranged in clusters, but binding of FIS occurs without cooperativity. The cooperative binding mode of LRP

often masks the primary binding site and leads to efficient binding of LRP not only

at the high-affinity site but includes multiple suboptimal sites. This explains the

extended regions of protection within the regulatory regions of many LRP-dependent

operons visible in footprint analyses (Wang and Calvo 1993; Nou et al. 1995; Pul

et al. 2007). Recent analyses have demonstrated that LRP can bind to non-specific

DNA targets with high affinity and in cooperative manner (Peterson et al. 2007).

Such cooperative extension after initial binding to a high affinity site appears to be

a common phenomenon for LRP-dependent transcription regulation and has speci­

fically been demonstrated in a phasing study, where a synthetic AT-rich sequence

had been fused at varying distance upstream of two differentially regulated promotes (Pul et al. 2008).Binding of LRP clearly has an impact on DNA structure.

The orientation of the helix-turn-helix-domains on the outside of the octameric

LRP core suggests a model in which the DNA is wrapped around the protein core

(Thaw et  al. 2006). This is consistent with footprint analyses, which for many

operons show that LRP-DNA interactions extend over a range of more than 100

base pairs. Moreover, the wrapping of DNA around LrpC has been visualized by

electron microscopy and AFM (Beloin et al. 2003). In addition, it has been found

that LRP binding results in a periodic pattern of DNase I or hydroxyl radical protected and hypersensitive sites (phased hypersensitivity), indicating LRP-dependent

bending or wrapping of the DNA (Stauffer and Stauffer 1994; Ferrario et al. 1995;

Nou et al. 1995; Pul et al. 2005; McFarland and Dorman 2008). For LrpC from B.

subtilis and E. coli LRP it has been shown that the formation of such higher-order

nucleoprotein complexes constrains supercoils (Beloin et al. 2003; Pul et al. 2007).

Interestingly, the reported direction of the constrained supercoils differs for LRP

and LrpC. Whether this difference is due to the different quarternary structures of

the two homolog proteins or reflects general differences in their function needs

further experimental clarification.

It should be noted at this point, that not all members of LRP family are global

regulators. H. influenzae LrfB shows 75% sequence identity with E. coli LRP but

has a specific regulatory role restricted to only a few genes (Friedberg et al. 2001).

This may be related to the lower expression level of LrfB of only 130 molecules per

cell. The authors conclude that the global regulatory function of LRP is restricted

to enteric bacteria, which encounter variable environmental conditions. It is likely,

8  Nucleoid-Associated Proteins: Structural Properties


therefore, that in each individual species the function of LRP as a global or a specific regulator is tuned to its expression level. In E. coli expression of the lrp gene

is inversely proportional to the growth rate and coupled to the quality of the media.

Moreover, it is induced directly or indirectly through positive regulation by ppGpp

(Landgraf et al. 1996). Transcription of the lrp gene is enhanced by relaxation of

the DNA template following inhibition of DNA gyrase (Müller et  al. 2009).

Mechanisms to regulate the cellular LRP concentration may thus determine whether

it acts as a global or a specific transcription factor.

8.5 IHF: Integration Host Factor

IHF was first described as the host factor required for integration of phage l in the

bacterial chromosome. Today it is known that IHF, in addition to its role in recombination or replication events, is involved in transcription regulation of more than

100 genes in E. coli and S. typhimurium (Arfin et al. 2000). The heterodimeric IHF

in E. coli is encoded by the highly homologous genes ihfA and ihfB (Weisberg

et al. 1996), which are translated to the approximately 10 kDa a-and b-subunits,


IHF binds specifically to the minor groove of DNA by recognizing the consensus sequence (A/T)ATCAANNNTT(G/A) (Hales et  al. 1994). This interaction

leads to the most pronounced characteristic of IHF, namely its ability to introduce

a sharp bend, almost a U-turn, into the DNA. This property makes IHF an important

architectural component for many DNA transaction reactions, which require bent

DNA conformations. As an example, the structure of E. coli IHF bound to the

H¢-site of phage l is shown in Fig. 8.4 (Rice et al. 1996). Each of the IHF protein

monomers is composed of three a-helices, forming the central body, and two

Fig. 8.4  Crystal structure of a DNA-IHF complex (PDB ID: 1IHF; (Rice et al. 1996)). The individual monomers (IHFa and IHFb) are indicated by a blue or green backbone, respectively. The two

b-ribbons wrap around the DNA through the minor grooves. The proline residues (shown in red)

interrupt the base stacking, leading to strong kinks in the DNA structure, which is shown in grey


Ü. Pul and R. Wagner

antiparallel b-ribbons, forming the IHF-arms. Each of the b-ribbon arms contains

a conserved proline residue close to the tip of the antiparallel strands, which, in the

bound complex, intercalates between adjacent DNA bases. The intercalation of the

two prolines abolishes the stacking interaction between the neighbouring bases,

which leads to a widening of the minor groove giving rise to two sharp kinks of the

target DNA. Stabilized by electrostatic interactions between positively charged

residues of the IHF body and the negatively charged DNA backbone the contour of

the DNA, deformed by the two kinks, results in a U-turn like bending of about

160°–180° (Rice et al. 1996).

A further remarkable aspect of IHF-DNA interaction resides in the sequencespecificity of IHF, which is rather uncommon for minor groove binding proteins.

IHF-dependent recognition of the DNA consensus-sequence involves contributions of both side chain-base contacts within the b-arms and also within the IHF

body. Thus, the interaction between IHF and DNA is a typical example for the

‘indirect readout’ mechanism for which the recognition and binding to the consensus sequence depends on the structural flexibility of the target DNA (Aeling

et al. 2006).

8.6 HU: Histone-Like Protein from Strain U93

The dimeric HU protein exhibits sequence and structural homology with the IHF

protein. The first high-resolution structure derived from B. stearothermophilus

depicts a dimeric molecule with two flexible basic arms, which fit into the minor

groove of DNA (White et al. 1999). In most bacteria HU exists as a homodimer.

In the enteric bacterium E. coli, however, it exists as heterodimer, with each subunit

being 9.5 kDa in size, encoded by the homologous hupA and hupB genes. The

subunits, HUa and HUb, show 70% homology to each other and approximately

35% homology to IHF. Despite the sequence and structural similarity between IHF

and HU, the latter protein binds sequence non-specific to the DNA but also bends

the target DNA through intercalation of two likewise conserved proline residues

between two base-pairs in the minor groove (Rice 1997; Bewley et  al. 1998;

Swinger et al. 2003) (Fig. 8.5). As in the case of IHF the intercalation of proline

residues introduces and stabilizes two kinks in the DNA. In a recent binding study

it was shown by FRET analysis that interaction of HU with a 34 bp DNA fragment

caused almost a 143° bending angle of the DNA (Koh et al. 2008). This result is

fully consistent with the resolved structure from co-crystals of Anabaena HU and

DNA shown in Fig. 8.5.

Consistent with its property to bend DNA HU plays an important architectural

role in all kinds of DNA transactions including transcription regulation. In combination with other regulators HU often facilitates or even enables the formation of

active DNA conformations through formation of higher-order nucleoprotein complexes. Such DNA structures often involve DNA loops as has been shown for

instance for the gal operon. In this case HU binds within the interoperator region

8  Nucleoid-Associated Proteins: Structural Properties


Fig.  8.5  Crystal structure of the Anabaena HU-DNA complex (PDB ID: 1P71; Swinger et  al.

2003). The individual monomers (HUa and HUb) are shown as blue or green backbones. The

structure resembles very much that for IHF. As shown for IHF two conserved proline residues at

the tip of the b-ribbons (coloured in red) intercalate between base pairs of the DNA minor groove,

inducing a strong DNA bend

of the gal operon facilitating the interaction of two bound GalR dimers, which lead

to a looped structure inadequate for transcription and therefore called repressosome

(Semsey et al. 2002). A similar role for HU has also been described for the sitespecific DNA inversion by the Hin recombinase. Here, the Hin-dependent assembly

of the invertasome is facilitated by HU, which enables the necessary DNA looping

(Haykinson and Johnson 1993) (Chapter 17).

Taking advantage of atomic force microscopy and magnetic tweezers technology

some crucial structural properties of HU-DNA complexes have been addressed.

Such studies indicated that HU, aside from inducing flexible bends in DNA, is able

to form rigid nucleoprotein filaments at higher HU concentrations, indicating the

existence of two different HU-DNA nucleoprotein complexes (Dame and Goosen

2002; van Noort et al. 2004). In such filament-like complexes HU is arranged helically

around the DNA (see also Fig. 8.7a).

Interestingly, the expression of the two homologous HU subunits is differentially

regulated in E. coli, leading to diverse subunit compositions of HU in the cell by

switching from predominantly HUa homodimers in early log phase to heterodimers at stationary phase (Claret and Rouvière-Yaniv 1997). This phase-dependent

expression pattern suggests that the different forms of HU are not functionally

equivalent. Rather the distinct dimeric forms exhibit a potential regulatory role in

response to different growth conditions (Claret and Rouvière-Yaniv 1997). The

ability to form distinct homo- or heterodimers among the NAP members is not

restricted to HU. A similar situation has been described for H-NS and the paralog

StpA. Both proteins, which are able to interact with each other, are also differentially regulated and disparate functions have been suggested for the distinct dimers

(Zhang et al. 1996). The implications of such homomeric or heteromeric complexes

between different NAP members for bacterial adaptation and pathogenesis have

been discussed in a previous review (Dorman et al. 1999).

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